Serum concentrations of 25-hydroxyvitamin D [25(OH)D] were determined for free-ranging and captive white-tailed deer (WTD). Effects of gender, season, and age on 25(OH)D concentrations were determined as well as comparisons to concentrations in serum from captive reindeer and elk. Seasonal variations in 25(OH)D concentrations were detected for both captive and free-ranging WTD with greatest concentrations detected in August/September (approximately 25 ng/mL) and lowest concentrations in February (approximately 5 - 10 ng/mL). Free-ranging WTD < 1 year of age had lower 25(OH)D concentrations (approximately 6 ng/mL) than did free-ranging WTD > 1 year of age (approximately12 ng/mL). For captive WTD fawns, 25(OH)D concentrations increased from 1 to 9 days of age (exceeding 100 ng/mL) and then steadily declined to approximately 10 ng/mL by 3 months of age. In general, differences in 25(OH)D concentrations based on gender were not detected. 25(OH)D concentrations in captive WTD did not differ from that of captive reindeer; yet, 25(OH)D concentrations were lower in WTD than in captive elk. Additional research is necessary to determine if low serum 25(OH)D concentrations during the winter or pre-weaning period are associated with increased rates of infectious and metabolic disease.