Peroxisome proliferators induce hepatic peroxisome proliferation and hepatic tumors in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including eicosanoids. Peroxisome proliferators transiently induce increased cell proliferation in vivo. However, peroxisome proliferators are weakly mitogenic and are not co-mitogenic with epidermal growth factor (EGF) in cultured hepatocytes. Earlier study found that the peroxisome proliferator ciprofibrate is comitogenic with eicosanoids. In order to study possible mechanisms of the comitogenicity of peroxisome proliferator ciprofibrate and eicosanoids, we hypothesized that the co-mitogenicity may result from synergistic or additive increases of second messengers in mitogenic signal pathways. We therefore examined the effect of the peroxisome proliferator ciprofibrate, prostaglandin F2 alpha (PGF2 alpha) and the combination of ciprofibrate and PGF2 alpha with or without growth factors on the protein kinase C (PKC) activity, and inositol-1, 4, 5-triphosphate (IP3) and intracellular calcium ([Ca2+]i) concentrations in cultured rat hepatocytes. The combination of ciprofibrate and PGF2 alpha significantly increased particulate PKC activity. The combination of ciprofibrate and PGF2 alpha also significantly increased EGF, transforming growth factor-alpha (TGF-alpha) and hepatic growth factor (HGF)-induced particulate PKC activity. The combination of ciprofibrate and PGF2 alpha greatly increased [Ca2+]i. However, the increases of PKC activity and [Ca2+]i by ciprofibrate and PGF2 alpha alone were much smaller. Neither ciprofibrate or PGF2 alpha alone nor the combination of ciprofibrate and PGF2 alpha significantly increased the formation of IP3. The combination of ciprofibrate and PGF2 alpha, however, blocked the inhibitory effect of TGF-beta on particulate PKC activity and formation of IP3 induced by EGF. These results show that co-mitogenicity of the peroxisome proliferator ciprofibrate and eicosanoids may result from the increase in particulate PKC activity and intracellular calcium concentration but not from the formation of IP3.