The triazine herbicides, atrazine (ATR), simazine (SIM), propazine (PRO), terbuthylazine (TBA), and their chlorinated metabolites have been implicated in the etiology of testicular dysgenesis by altering steroidogenesis. To further investigate their effects on testosterone biosynthesis, BLTK1 cells were used to evaluate steroid hormone levels and genome-wide gene expression. BLTK1 cells are a novel murine Leydig cell line possessing an intact steroidogenic pathway with constitutive low basal testosterone (T) levels that can be induced by recombinant human chorionic gonadotropin (rhCG). Triazines (ATR, SIM, PRO, and TBA) and their chlorometabolites (DEA, DIA, and DACT) induced concentration-dependent (1, 3, 10, 30, 100, 300, and 600 µM) increases in progesterone (P) and T levels relative to solvent control at 24h. Temporal analysis (300 µM at 1, 2, 4, 8, 12, 24, or 48h) elicited comparable P and T profiles by all compounds with varying efficacies (ATR > TBA > PRO > DEA > DIA > DACT >> SIM) that were similar to rhCG. ATR and TBA elicited time- and concentration-dependent induction of Star, Hsd3b6, and Hsd17b3 mRNA levels, whereas Hsd3b1, Cyp17a1, and Srd5a1 mRNA expression was repressed. PRO elicited similar albeit weaker effects, whereas SIM had negligible effects consistent with their induction of P and T levels. Whole-genome microarrays identified 797 differentially regulated genes elicited by 300 µM ATR, occurring primarily at later time points (> 12h) with overrepresented functions associated with steroidogenesis and cholesterol metabolism. These results indicate that changes in P and T levels can be partially attributed to triazine-elicited alterations in steroidogenic gene expression.