The human mast cell lines HMC-1560 and HMC-1560,816 were used to study histamine release, Ca2+ signaling and protein kinase C (PKC) localization and expression, with phorbol 12-myristate 13-acetate (PMA). Both sublines carry activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit tyrosine kinase activation. Both have the Gly-560 → Val mutation but only the second carries the Asp-816 → Val mutation. In this study, it was observed that the stimulation of PKC has different effects in HMC-1560 and HMC-1560,816 and this would be related to the difference in activating mutations in both mast cell lines. PKC activation increases ionomycin-induced histamine release in HMC-1560. This article demonstrates an opposite histamine response in HMC-1560,816 cells, even though classical PKCs are the family of isozymes responsible for this effect in both cellular lines. Furthermore, it can be observed that upon cell stimulation with PMA, primarily cytosolic PKC translocates to the nucleous in HMC-1560,816 cells, but not in HMC-1560 cell line. J. Cell. Biochem. 112: 2637–2651, 2011. © 2011 Wiley-Liss, Inc.