Results of in vivo studies indicate dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts. It was hypothesized that both NCG and ARG will alter hormone-induced estradiol (E2) production by granulosa cells (GC), explaining why these compounds could improve reproductive performance in pigs. The objective of these studies, therefore, was to evaluate the direct effects of NCG and ARG on porcine GC proliferation and steroidogenesis, using an in vitro cell culture system. The GC from small (SM; 1-5 mm) and large (LG; >5 mm) pig follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/mL of testosterone (as an E2 precursor), and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/mL), insulin-like growth factor-1 (IGF1; 30 ng/mL), or both. Numbers of GC were determined at the end of the experiment and concentrations of progesterone (P4) and E2 in culture medium were determined. Results indicated that LG-follicle GC were more responsive to NCG and ARG than SM-follicle GC. Specifically, in LG-follicle GC, NCG inhibited (P < 0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. In SM-follicle GC, treatment with NCG and ARG decreased IGF1 plus FSH induced P4 production, but E2 production and cell proliferation were not affected. These studies indicate that NCG and ARG may directly affect follicular function in pigs.