We explore the interaction of muscle innervation and gonadal hor- mone action in the pubococcygeus muscle (Pcm) after castration and hor- mone replacement. Male Wistar rats were castrated and the Pcm was unilaterally denervated; after 2 or 6 weeks, the cross-sectional area (CSA) of Pcm fibers was assessed. Additional groups of castrated rats were used to examine the effects of hormone replacement. At 2 weeks post surgeries, rats were implanted with Silastic capsules containing either dihydrotes- tosterone (DHT), estradiol benzoate (EB) or both hormones, and the CSA of Pcm fibers was assessed after 4 weeks of hormone treatment. At 2 weeks post surgeries, gonadectomy without hormone replacement resulted in reductions in the CSA of Pcm fibers, and denervation com- bined with castration increased the magnitude of this effect; further reductions in CSA were present at 6 weeks post surgeries, but again denervation combined with castration increased the magnitude of this effect. Hormone replacement with DHT resulted in hypertrophy in the CSA of nondenervated muscles compared to those of intact normal males, but this effect was attenuated in denervated muscles. Hormone replace- ment with EB treatment prevented further castration-induced reductions in CSA of nondenervated muscles, but denervation prevented this effect. Similar to that seen with treatment with EB alone, combined treatment with both DHT and EB prevented further reductions in CSA of Pcm fibers in nondenervated muscles, but again denervation attenuated this effect. Thus, while hormone replacement can reverse or prevent further castration-induced atrophy of Pcm fibers, these effects are depen- dent on muscle innervation. Anat Rec, 300:1327–1335, 2017. VC 2017 Wiley Periodicals, Inc.