OBJECTIVE To identify factors (including exposure to specific antiretroviral drugs) associated with severe vitamin D deficiency (VDD) in HIV-infected individuals and to explore the effects of severe VDD and antiretroviral drug exposure on serum alkaline phosphatase (ALP) as surrogate marker of bone turnover. METHODS Cross-sectional survey of vitamin D status among HIV-infected patients attending for routine clinical care at a large London HIV clinic. METHODS Severe VDD was defined as 25(OH)D levels of less than 10 microg/l (<25 nmol/l). Multivariate logistic regression analysis was used to identify factors associated with severe VDD and upper quartile ALP levels. RESULTS Vitamin D levels were measured in 1077 patients and found to be suboptimal in 91%. One-third of patients had severe VDD. Black ethnicity, sampling in winter, nadir CD4 cell count less than 200 cells/microl, and exposure to combination antiretroviral therapy were associated with severe VDD. In analyses restricted to patients on combination antiretroviral therapy, current efavirenz use was significantly associated with severe VDD [adjusted odds ratio 2.0 (95% confidence interval 1.5-2.7)]. Current tenofovir [adjusted odds ratio 3.5 (95% confidence interval 2.3-5.2)] and efavirenz use [adjusted odds ratio 1.6 (95% confidence interval 1.02-2.4)], but not severe VDD [odds ratio 1.1 (0.8-1.5)], were associated with increased bone turnover (upper quartile ALP). CONCLUSIONS Efavirenz was associated with severe VDD, a condition associated with multiple adverse health outcomes, and efavirenz and tenofovir with increased ALP. The clinical significance of these findings requires further investigation, given the widespread use of efavirenz and tenofovir in first-line combination antiretroviral therapy.