A role for estrogen receptor (ER)-alpha in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ERalpha(-/-) mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ERalpha(-/-) mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ERalpha and FGF10. ERalpha was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ERalpha or ERbeta with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ERalpha agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3-5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ERalpha is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ERalpha can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.