It is well recognized that high-dose estrogen induces a marked osteogenic response in long bones of female mice. In light of evidence which suggests that nitric oxide synthase (NOS) plays a role in regulation of osteoblast activity, we analyzed whether NOS is involved in mediating this response. Intact female mice were administered 17beta-estradiol (E(2)) either alone or in combination with N(G)-nitro-L-arginine methylester (L-NAME) or aminoguanidine (AG), over 24 days. The former inhibits both constitutive and inducible isoforms of NOS, whereas the latter is a selective inhibitor of inducible NOS. Bone mineral density (BMD) of the femur was subsequently measured by dual-energy X-ray absorptiometry (DXA), and histomorphometry performed at the proximal metaphysis on longitudinal tibial sections. As expected, E(2) given alone led to a marked accumulation of cancellous bone at the proximal tibial metaphysis, associated with a significant gain in femoral BMD, and an increase in cancellous mineralizing surfaces as assessed by histomorphometry. Neither L-NAME nor AG affected cancellous histomorphometric indices when given alone. However, when administered in combination with L-NAME, the magnitude of the skeletal response to E(2) was significantly reduced. The tendency for L-NAME to reduce estrogen-induced bone formation within the proximal tibial metaphysis was more marked distally compared with proximally. In contrast, AG showed no tendency to suppress the osteogenic response to E(2). Subsequently, we examined the effect of E(2) administration on expression within mouse femoral bone marrow of endothelial NOS (eNOS), which is the predominant constitutive isoform of NOS within bone. No change in eNOS mRNA levels was observed following E(2) administration, as assessed by reverse transcription-polymerase chain reaction (RT-PCR). Taken together, our results suggest that eNOS plays a role in mediating estrogen-induced bone formation in intact female mice, possibly as a consequence of posttranscriptional regulation of eNOS activity by estrogen.