Recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) effects on basal, insulin-like growth factor I (IGF-I)-, and follicle-stimulating hormone (FSH)-stimulated progesterone (P4) secretion and [3H]aminoisobutyric acid (AIB) uptake by primary porcine granulosa cells (MDGs) and MDGs that have been passaged once (MDGp1) were assessed. Cells were treated concurrently or were preincubated with rhIGFBP-3 followed by treatment. rhIGFBP-3 had no effect on MDG or MDGp1 cell numbers after 24 h. Cotreatment with rhIGFBP-3 inhibited P4 secretion after treatment with FSH, IGF-I, and FSH plus IGF-I. FSH did not stimulate [3H]AIB uptake. However, the IGF-I-stimulated increase in [3H]AIB uptake was completely prevented by concurrent treatment with IGFBP-3. Preincubation of MDGp1 cells with IGFBP-3 dose dependently inhibited FSH- and IGF-I-stimulated P4 secretion. This inhibition was associated with increased cell association of the binding protein and increased IGF-I binding to the cells. These results indicate that IGFBP-3 is inhibitory to a variety of crucial functions in porcine granulosa cells, supporting a role for it in the regulation of granulosa cell function.