In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.