Previous studies have reported the roles of Ca(2+) in steroidogenesis. The present study has investigated an inhibitory effect of Ca(2+) influx through L-type Ca(2+) channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca(2+) influx through L-type Ca(2+) channels using the selective Ca(2+) channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca(2+) channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca(2+) influx through L-type Ca(2+) channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca(2+) channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca(2+) channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca(2+) channel blockers are able to induce Star gene expression. These observations indicate that Ca(2+) influx through L-type Ca(2+) channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca(2+) channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.