The murine mast cell line PB-3c is dependent on interleukin 3 (IL-3) with respect to survival and proliferation. These cells also require IL-3 to display antigen-mediated serotonin release, which is coupled to a transient increase of cytosolic free calcium ([Ca2+]i). The antigen-mediated exocytosis is inhibited by phorbol 12-tetradecanoate 13-acetate (PTA), an activator of phospholipid/Ca2+-sensitive protein kinase. In contrast, the malignant mast cell variant PB-1 is IL-3 independent with respect to proliferation but is unable to undergo antigen-mediated exocytosis. Yet this cell line exhibits basal levels of [Ca2+]i, serotonin content, and numbers of IgE receptors comparable to those of PB-3c cells. Subcellular distribution studies revealed that the specific activity of cytosolic protein kinase C of PB-1 cells was only 40% of that found in PB-3c cells. Furthermore, the PB-1 cells showed a significantly higher specific activity of membrane-bound protein kinase C than PB-3c cells. Scatchard plot analysis of [3H]-phorbol 12,13-dibutyrate binding to intact PB-1 cells demonstrated the presence of 20% high-affinity (Kd = 6 nM) and 80% low-affinity (Kd = 60 nM) phorbol ester receptors, whereas PB-3c cells displayed only the low-affinity phorbol ester binding. Immunological characterization of protein kinase C from both cell lines revealed the presence of a normal 77-kDa protein kinase C holoenzyme in both cell lines. In addition, a 72-kDa protein kinase C-related protein band was found mainly in the membrane fraction of the PB-1 variant. It is suggested that this altered and membrane-bound form of protein kinase C may be involved in the blockage of the antigen-mediated exocytosis of PB-1 cells.