To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.