Abstract Objective: Evidence from randomised controlled trials suggests that vitamin D may reduce multimorbidity, but very few studies have investigated specific determinants of vitamin D2 and D3 (two isoforms of 25-hydroxyvitamin D). The aim of the study was to investigate the determinants of vitamin D2 and D3 and to identify the risk factors associated with hypovitaminosis D. Design: Cross-sectional study. Setting: Northern Finland Birth Cohort 1966. Participants: 2374 male and 2384 female participants with data on serum 25(OH)D₂ and 25(OH)D₃ concentrations measured at 31 years of age (1997), together with comprehensive measures of daylight, anthropometric, social, lifestyle and contraceptive cofactors. Methods: We assessed a wide range of potential determinants prior to a nationwide fortification programme introduced in Finland. The determinants of 25(OH)D₂, 25(OH)D₃ and 25(OH)D concentrations were analysed by linear regression and risk factors for being in lower tertile of 25(OH)D concentration by ordinal logistic regression. Results: At the time of sampling, 72% of the participants were vitamin D sufficient (≥50 nmol/L). Low sunlight exposure period (vs high) was associated positively with 25(OH)D₂ and negatively with 25(OH)D₃ concentrations. Use of oral contraceptives (vs nonusers) was associated with an increase of 0.17 nmol/L (95% CI 0.08 to 0.27) and 0.48 nmol/L (95% CI 0.41 to 0.56) in 25(OH)D₂ and 25(OH)D₃ concentrations. Sex, season, latitude, alcohol consumption and physical activity were the factors most strongly associated with 25(OH)D concentration. Risk factors for low vitamin D status were low sunlight exposure defined by time of sampling, residing in northern latitudes, obesity, higher waist circumference, low physical activity and unhealthy diet. Conclusions: We demonstrate some differential associations of environmental and lifestyle factors with 25(OH)D₂ and 25(OH)D₃ raising important questions related to personalised healthcare. Future strategies could implement lifestyle modification and supplementation to improve vitamin D2 and D3 status, accounting for seasonal, lifestyle, metabolic and endocrine status.