To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. While stimulation with 0.05 mM dibutyryl cyclic AMP (Bt₂cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 µM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7 fold and 34.3 fold, respectively. In the presence of indomethacin, the level of Bt₂cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. While SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt₂cAMP-stimulated StAR protein expression and steroid production. Over-expression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.