Relaxin (RLX) is a product of the theca interna layer of the developing pig follicle. However, its physiological role within the ovary is unknown. The purpose of this study was to determine the effects of RLX alone or in conjunction with insulin and insulin-like growth factor-I (IGF-I) on porcine granulosa cell (GC) DNA synthesis and proliferation in vitro. The results showed RLX (1-1000 ng/ml), insulin (1-1000 ng/ml), and IGF-I (1-30 ng/ml) alone stimulated [3H]thymidine incorporation into DNA by GC from small, medium, and large porcine follicles (P < 0.05). The effect of RLX in combination with insulin or with IGF-I on DNA synthesis by GC from small and medium follicles was greater than the effect of either single factor (P < 0.05). In the large follicle, RLX increased GC DNA synthesis synergistically with insulin and IGF-I. FSH (100 ng/ml) or estradiol (10(-7) M) had no effect on basal or RLX-induced [3H]thymidine incorporation into GC DNA at any stage of follicular development. After 6 days incubation in vitro, RLX dose-dependently increased proliferation of GC from small follicles (P < 0.05). The increased cell number in response to RLX correlates with RLX-induced [3H]thymidine incorporation into DNA by these GC. Hydroxyurea, an inhibitor of DNA synthesis, blocked RLX-induced thymidine incorporation, indicating that the increase in thymidine incorporation in response to RLX was due to DNA synthesis and not repair. This is the first demonstration that RLX acts as a growth factor in the porcine follicle by increasing GC DNA synthesis and proliferation in vitro. RLX enhanced the growth-promoting effects of insulin and IGF-I on GC, which suggests an interaction of RLX with these proteins during follicular growth. These studies support the concept that locally produced thecal RLX alone or in combination with other growth effectors may play a key role in GC growth during porcine follicular development.