The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.