The nonsteroidal anti-inflammatory drug (NSAID) diclofenac (DF) is associated with idiosyncratic hepatotoxicity and several other distinct hypersensitivity reactions. The mechanism(s) are unknown but evidence suggests both cell-mediated and antibody-mediated immune effector systems may be involved. In the present studies, the immunostimulating potential of DF was evaluated using the direct and TNP-Ficoll (trinitrophenyl (TNP)-Ficoll) popliteal lymph node assays (PLNA). These assays were conducted in naive mice, T-cell-deficient mice, or in mice that had been pretreated with a single oral dose of DF. In naive mice, DF induced a dose-, and time-dependent reaction in the direct PLNA. A significant increase in popliteal lymph node (PLN) weight and PLN cellularity was detected 7 days after the injection of 0.50 and 0.75 mg DF, whereas 0.25 mg DF produced no observable effect. With 0.75 mg, there was a rapid accumulation of cells in the PLN between days 5 and 6, with maximum PLN cellularity observed between days 7 and 10. The immunostimulating effects of DF were significantly attenuated in T-cell-deficient mice. In the TNP-Ficoll PLNA conducted in naive mice, DF caused a dose-dependent increase in PLN cellularity on day 7 with a time-dependent increase in anti-TNP antibody forming cells (AFCs) in the PLN; the reaction was dominated by IgM anti-TNP AFCs from day 4 through day 7, but IgG1 anti-TNP AFCs and IgG3 anti-TNP AFCs were detected beginning on day 5 and day 6, respectively. Relative to mice pretreated with vehicle (ddH2O), mice orally pretreated with DF had a significantly greater increase in PLN weight 5 days following the injection of 0.25 mg DF and a significantly greater increase in PLN weight and cellularity 4 days following the injection of 0.50 mg DF. Oral pretreatment with DF had no observable effect on the direct PLN reaction induced following the footpad injection of the irrelevant drugs, D-penicillamine (D-PEN) or streptozotocin. When 0.50 mg DF was co-injected with TNP-Ficoll, mice orally pretreated with DF, compared to vehicle-pretreated mice, and had a significantly greater increase in IgM anti-TNP AFCs on day 4, and a significant increase in both IgG1 and IgG3 anti-TNP AFCs on day 7. Additionally, IgG1 anti-TNP AFCs were detected in the PLN of DF-pretreated mice as early as day 4. No differences in anti-TNP AFCs were detected when orally pretreated mice were injected with 0.50 mg D-PEN. Collectively, these results demonstrated that DF (i) is an immunostimulating drug that induced a dose-, time- and T-cell-dependent PLN reaction in naive mice, (ii) provided non-cognate help that produced antibody against co-injected TNP-Ficoll, and (iii) mice orally pretreated with DF had DF-specific increased responsiveness in the direct PLNA, which (iv) resulted in accelerated and augmented AFC production against co-injected TNP-Ficoll. These novel findings suggest that oral administration of DF may result in primed T cells that respond with footpad injection. Thus, the oral pretreatment modification of the PLNA should be further explored as a possible alternative to hypersensitivity testing with drugs administered via the oral route. Additional studies with other compounds known to produce hypersensitivity reactions are needed.