Laboratory and epidemiological data have linked vitamin D to breast cancer prevention. Beside dietary intake, endogenous production of vitamin D substantially contributes to a subject's vitamin D status. Most studies, however, have assessed dietary intake only. Although differential effects of vitamin D on premenopausal and postmenopausal breast cancer have been discussed, this is the first study to investigate the association of plasma 25-hydroxyvitamin D [25(OH)D], as indicator of the overall vitamin D status, with breast cancer risk with restriction to premenopausal women only. We used data of a population-based case-control study comprising 289 cases and 595 matched controls. Information on sociodemographic and breast cancer risk factors was collected by questionnaire and plasma 25(OH)D was measured by enzyme immunoassay. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. We observed a significant inverse association between breast cancer risk and plasma 25(OH)D concentrations. Compared with the lowest category (<30 nmol/L), the ORs (95% CI) for the upper categories (30-45, 45-60, >or=60 nmol/L) were 0.68 (0.43-1.07), 0.59 (0.37-0.94) and 0.45 (0.29-0.70), respectively (p(trend) = 0.0006). The association was shown to be nonlinear (p(nonlinearity) = 0.06) in fractional polynomial analysis with a stronger effect in women at low plasma 25(OH)D levels, providing some evidence of a threshold effect (at circa 50 nmol/L). The association was stronger in progesterone receptor negative tumors, with suggestive evidence of effect heterogeneity (p(heterogeneity) = 0.05, case-only model). Our findings support a protective effect of vitamin D for premenopausal breast cancer.