Background: Gap-junctional intercellular communication (GJIC) is implicated in physicological processes and it is vitally important for granulosa cell (GC) differentiation and oocyte growth. We investigated the expression of connexin 43 (Cx43), a gap junctional protein, in normal and androstenedione-induced polycystic ovary (PCO), the effects of androstenedione on Cx43 expression, GJIC and progesterone production in granulosa cells in vitro. Methods: Isolated GCs from rat ovary were supplemented with FSH and dripped with EHS-matrix (EHS-drip) in culture media, were treated with physiological (10-7 M) or pathological (10-5 M) androstenedione concentrations to induce differentiation. Cx43 protein levels were assessed by Western blotting. Immunohistochemistry was also used to determine the localization of Cx43 in GCs and corpus luteum (CL) of controls and PCOs. Differentiation of GCs was determined by progesterone assay and Lucifer yellow dye transfer for GJIC status. The degree of significance of variations between the results was analyzed by ANOVA using SPSS (version 11.5; 2002). Results: Cx43 localized in the GC layer of both the control and PCOs. Its protein levels were upregulated in PCO rat ovaries. GCs in culture with or without androstenedione had a punctate membranous distribution of Cx43. However, androstenedione increased GJIC and upregulated progesterone and Cx43 protein levels. Inhibiting GJIC by 18- GA in androstenedione-treated GCs caused partial inhibition of progesterone production, suggesting a possible role of GJIC in mediating the action of androstenedione on GC differentiation. Conclusion: This study presented a suitable culture model for polycystic ovary syndrome and showed that Cx43 and GJIC might contribute to the pathogenesis of polycystic ovary syndrome.