Nitric oxide (NO) has been implicated in bone growth and remodeling by studies showing that inhibition of NO‐synthase (NOS) activity retards normal gain in bone mineral density both during skeletal development and after sexual maturity. In the present study, we aimed to assess the level of expression and cellular localization of the three NOS isoforms during skeletal bone development from neonatal to sexual maturity in female Wistar rats. Reverse transcription polymerase chain reaction (RT‐PCR) was used to analyze the presence of NOS1 (neuronal), NOS2 (inducible), and NOS3 (endothelial) transcripts in femoral bone from neonatal, 4‐, 8‐, and 12‐week‐old rats. RT‐PCR amplified NOS1, NOS2, and NOS3 transcripts of 472‐, 807‐, and 289‐bp, respectively. There were no detectable differences in the levels of NOS1 mRNA between the groups; however, NOS2 mRNA was more abundant in the neonatal group compared with 4‐, 8‐, and 12‐week groups. Expression of NOS1 protein could not be detected in bones by either Western blotting or immunocytochemistry in any of the age groups investigated. Western blots for NOS2 revealed expression in the neonatal group only and it was not detected in any of the older age groups. Immunostaining for NOS2 was also most evident in the neonatal group and was localized specifically to trabecular osteoblasts and osteoclasts. In all age groups studied, NOS3 mRNA and protein were found in bone‐resorbing osteoclasts, cuboidal active osteoblasts, and osteocytes. Semiquantitative RT‐PCR provided evidence of down‐regulation of NOS3 transcripts during the skeletal development. This was confirmed using in situ hybridization, which showed higher expression in neonatal and 4‐week groups than in other groups. Western blots and counting the ratio of trabecular osteoblasts that were NOS3 immunoreactive showed parallel down‐regulation of NOS3 protein during skeletal development. Taken together, these data show that there is regulation of NOS2 and in particular NOS3 expression during skeletal development and this may be significant to trabecular bone growth and remodeling.