BACKGROUND: Decreased 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with an increased prevalence and severity of asthma and a lower response to inhaled corticosteroids. OBJECTIVE: The objective was to determine the association between serum 25(OH)D concentrations and asthma prevalence, severity, and response to asthma treatment. DESIGN: Secondary analyses were conducted in 2 samples of adolescents 12-20 y of age: 1) NHANES 2001-2006 (n = 6487), a cross-sectional nationally representative sample of the US population, and 2) a cohort of inner-city adolescents with asthma managed prospectively for 46 wk with guidelines-based therapy in the Asthma Control Evaluation (ACE; n = 226) trial. RESULTS: Mean (+/-SD) serum 25(OH)D concentrations in the NHANES and ACE samples were lower in African Americans than in non-African Americans (NHANES: 14.9 +/- 6.5 compared with 23.0 +/- 8.4 ng/mL, P < 0.0001; ACE: 11.2 +/- 6.9 compared with 15.8 +/- 7.1 ng/mL, P < 0.0001). In the NHANES sample, mean concentrations did not differ between participants without and with asthma (African Americans: 14.9 +/- 6.4 compared with 15.0 +/- 6.6 ng/mL, respectively, P = 0.87; non-African Americans: 23.0 +/- 8.5 compared with 23.6 +/- 8.2 ng/mL, respectively, P = 0.16). In the ACE models that used either a predefined cutoff (<20 ng/mL) or linear regression, 25(OH)D concentrations showed either no relation or minor contradictory correlations with indicators of asthma severity, treatment requirements, spirometry, or atopy/inflammation. CONCLUSION: In 2 samples of adolescents, overall serum 25(OH)D concentrations were low and were not consistently associated with the presence of asthma, multiple asthma characteristics, asthma morbidity, or response to treatment. The ACE trial was registered at clinicaltrials.gov as NCT0011441.