INTRODUCTION The aim of the present study was to assess the prevalence of vitamin D deficiency and toxicity, the frequency of 25-hydroxyvitamin D (25[OH] D) testing, and 25(OH) D variations with respect to patient gender, patient age and season in New South Wales, Australia. METHODS A retrospective analysis of pathology records was performed to ascertain patient age, patient gender, sample collection date, plasma or serum 25(OH) D levels, calcium and parathyroid hormone (PTH) levels, and test numbers between 2001 and 2010. Linear regression with Bonferroni correction was used to calculate and compare ageadjusted mean 25(OH) D levels. Relationships of 25(OH) D with PTH and calcium were tested using Spearman's rank correlation. RESULTS 25(OH) D testing increased by 730% over the ten-year study period. In 2010, many men (33%) and women (40%) were, to some degree, vitamin D deficient (<= 50 nmol/L). Vitamin D toxicity was rare, with only one instance noted. 25(OH) D levels correlated positively with calcium and negatively with PTH levels. 25(OH) D levels decreased with age. In 2010, 25(OH) D levels were highest in February and lowest in September/October. Cyclical variation was observed for 25(OH) D levels between 2006 and 2010. CONCLUSION We found that vitamin D deficiency was prevalent in both men and women, with a higher prevalence in the latter, despite the substantial increased demand for 25(OH) D testing in our population over the decade. Vitamin D deficiency was associated with elevated PTH levels. Vitamin D toxicity was rare and only observed once during our study period. 25(OH) D levels decreased with age and varied with season, with the highest levels observed in late summer and the lowest in early spring.