Serum levels of 25-hydroxyvitamin D are important in establishing true vitamin D levels in humans. The purposes of this study were to develop a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of 25-hydroxyvitamin D(2) and D(3) and establish reference intervals for these analytes. Chromatographic separation of 25(OH)D(2) and 25(OH)D(3) was achieved after adding deuterated Delta(9)-tetrahydrocannabinol (D(9)-THC-D(3)) and organic extraction. The 3 ions were ionized using positive electrospray ionization and detected in the multiple-reaction monitoring mode using mass (m)/charge (z) transitions of 318.15 > 196.20 (Delta(9)-THC-D(3)), 401.15 > 365.2 (25(OH)D(3)), and 413.15 > 355.20 (25(OH)D(2)). Reference interval study results were compared with current 25(OH)D recommendations. Elution of 25(OH)D(2), 25(OH)D(3), and Delta(9)-THC-D(3) was achieved after 3.0 minutes (total run time, 6.0 minutes). Within- and between-run coefficients of variation were less than 11%. Deming regression of radioimmunoassay and LC-MS/MS methods for total 25(OH)D levels yielded a slope of 0.97 (95% confidence interval, 0.88-1.05) and y-intercept of -1.74 ng/mL. Reference intervals were less than recommended levels (D(2), 0.0-12.1; D(3), 5.5-41.4; total vitamin D, 6.0-43.5 ng/mL [0-30, 14-103, 15-109 nmol/L, respectively]) with no statistically significant differences in race, age, or sex. This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 25(OH)D(2) and 25(OH)D(3) at nanomolar concentrations.