Objective: To further investigate the action mechanisms of berberine (Ber), and assess the effects of Ber on the in vitro formation of cGMP and cAMP in the isolated rabbit corpus cavernosum. Methods: Isolated segments of the rabbit corpus cavernosum were exposed to different concentrations of Ber, and the dosage-dependent accumulations of cGMP and cAMP were determined in the tissue samples by means of ~ 125I radioimmunoassay. Responses of the isolated tissue preparations to Ber were compared with those obtained with the reference compound sildenafil (Sil). Results: Ber increased cGMP concentrations directly (P< 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both Ber and Sil increased cGMP with increasing dosage (P< 0.01), the EC 50 values being 1.32 and 0.67 μmol/L respectively. With the same concentration, neither Ber nor Sil influenced the cAMP level significantly (P> 0.05). In the presence of PGE 1, a stimulator of cAMP, Ber and Sil also raised the cAMP level concentration (P< 0.01), the EC 50 values being 4.90 (Ber) and 6.53 (Sil) μmol/L respectively. Conclusion: Ber can increase cGMP and cAMP concentrations in the corpus cavernosum smooth muscles, which may contribute to its action of relaxing corpus cavernosum smooth muscles.