Low vitamin A and carotenoid levels could increase the risk of sexual HIV acquisition by altering the integrity of the genital epithelium or by immunologic dysfunction. We addressed this issue by measuring serum vitamin A and carotenoid levels in patients who were at risk of subsequent HIV infection. In a nested case-control study in individuals attending two sexually transmitted disease (STD) clinics in Pune, India, serum micronutrient levels were measured in 44 cases with documented HIV seroconversion (11 women and 33 men) and in STD patients matched for gender and length of follow-up with no subsequent HIV seroconversion (controls). STD patients in Pune had low vitamin A and carotenoid levels, and low serum beta-carotene levels were independently associated with an increased risk of subsequent HIV seroconversion. STD patients with beta-carotene levels less than 0.075 micromol/L were 21 times more likely to acquire HIV infection than those with higher levels (adjusted odds ratio = 21.1; p =.01). No such association was observed in case of other non-provitamin A carotenoids. This study reports the first evidence of an association between low serum provitamin A carotenoid levels and an increased risk for heterosexual HIV acquisition in STD patients in Pune, India.