Androgens are essential and sufficient for prostate gland morphogenesis, however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone (T) on the expression of several critical prostate morphoregulatory genes. Rat ventral (VP) and lateral (LP) prostates were collected at birth and contralateral lobes were cultured for 18 hr in the presence or absence of 10nM T with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. While expression of Fgf10, Nkx3.1 and Ptc was increased in both prostate lobes, other genes were regulated by T in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Since previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.