There have been few epidemiologic studies on vitamin D status in Asians. The purpose of this study was to investigate the 25-hydroxyvitamin D (25[OH]D) levels in active women of middle and advanced age in Japan. We targeted 236 women who participated in an annual health check-up program in September 1997. Among them, 160 women were examined. Serum 25(OH)D2 and 25(OH)D3 levels were determined with high-performance liquid chromatography. In addition, the study included survey questions regarding age, body weight and height, occupation, use of skin protection, clinical and reproductive histories, and lifestyles. The average age was 65.6 y (SD = 8.3). The mean concentrations of 25(OH)D2 and 25(OH)D3 were 0.5 (SD = 3.2) nmol/L and 78.3 (SD = 17.8) nmol/L, respectively. None of the subjects showed hypovitaminosis D (25[OH]D < 37.0 nmol/L). Concentrations of 25(OH)D3 did not change with age (r = -0.079, P = 0.32), nor did other variables associated with 25(OH)D3 concentrations except for "engaging in farming" (P = 0.03) in the occupational category. These findings indicate that 25(OH)D levels in active middle- and advanced-aged women in Japan have appropriate vitamin-D status. Studies to elucidate and assess the dietary intake of vitamin D in Japanese women can be of further benefit.