Phenylazo-naphthol (PAN) allergy induces visibly well-defined and late-appearing hyperpigmentation of brownish yellow guinea pig skin in clear contrast to dinitrochlorobenzene (DNCB) allergy, which has very low incidence of hyperpigmentation. Skin extract from PAN allergy at 20-29 d post-challenge exhibited marked melanogenic stimulatory effects (3H2O release and 14C-thiouracil incorporation) when added to cultured guinea pig melanocytes. The time course in the appearance of melanogenic factor was definitely consistent with the induction pattern of visible pigmentation. By contrast, the addition of DNCB-challenged skin extract demonstrated no significant stimulating effect on melanogenesis in either assay system on any of the post-challenge days tested. Assay of intracellular inositol 1,4,5-trisphosphate formed through incubation with the melanocytes demonstrated that the PAN-allergy skin extract at day 28, which contains definite melanogenic factors, stimulated the formation of inositol 1,4,5-trisphosphate that occurs around 50 seconds in contrast to no or little increase with extracts obtained at days 0 and 1 post-challenge. Gel chromatographic analysis revealed that the PAN-allergy skin extract at day 28 contained a newly generated melanogenic fraction with a molecular weight of approximately 9000 Da which was also capable of stimulating DNA synthesis and activating the signal-transduction process (inositol trisphosphate formation) when added to guinea pig melanocytes. Both stimulations of melanogenesis and DNA synthesis by the 9000 Da fraction were completely abolished by the prior and simultaneous addition of protein kinase C (PKC) inhibitor (H-7) or its down-regulatory agent, phorbol 12,13-dibutyrate (PdBu). Taken together, these results suggest that PAN allergy provides a new mechanism of hypermelanization in which endogenous factors synthesized within skin induce the activation of signal-transduction pathways such as phosphoinositide turnover through ligands-receptor binding, resulting in the stimulation of melanocytes possibly through the activation of PKC.