Serum fluoride in relation to the prevalence of skeletal fluorosis was investigated in two villages in Jiangsu Province, China. In the high-fluoride village of Wamiao, 132 adults (average age 52.36 years; water fluoride 2.18±0.86mg/L; range 0.85–4.50mg/L) were surveyed. In the low-fluoride village of Xinhuai, 35 adults (average age 48.11 years; water fluoride 0.37±0.09 mg/L; range 0.21–0.55mg/L) were surveyed. Subjects were recruited by sampling according to the fluoride content of the drinking water in their household wells. When the subjects were divided into five subgroups according to their serum fluoride concentration, higher serum fluoride concentration was strongly associated with a higher prevalence of skeletal fluorosis in the form of a significant positive dose-response relationship (regression equation: Y = –27.29+890.42X–223.20X2). In Wamiao village a significant difference was also found between serum fluoride concentrations in 41 subjects with X-ray detectable skeletal fluorosis and in 91 subjects without X-ray detectable skeletal fluorosis. Gender related differences in serum fluoride concentration, household well water fluoride, and the prevalence of skeletal fluorosis were not found in the subjects in Wamiao village. These findings indicate that serum fluoride concentrations have a significant positive dose-response relationship with the prevalence of skeletal fluorosis in an endemic fluorosis area associated with high-fluoride drinking water.