Free holo-retinol binding protein (RBP) [i.e., unbound to transthyretin (TTR)] plays a role in transporting vitamin A across the placenta during pregnancy. In a cross-sectional study of clinically healthy urban women, we assessed the association among clinical and biochemical factors on estimated concentrations of free holo-RBP during the last trimester of pregnancy. Serum samples obtained from a subsample of women (n = 259), who had participated in the Night Vision Threshold Test study in Nepal, were analyzed for determinations of retinol by HPLC, and RBP, TTR, and alpha-1 acid glycoprotein by radial immunodiffusion. Free holo-RBP concentrations were calculated using dissociation constants for free holo- and apo-RBP. Among these women, 30% were vitamin A deficient based on either the RBP:TTR index < or = 0.36 or serum retinol < 1.05 micromol/L. Using stepwise regression analyses, the RBP:TTR index explained 75% of the variance in free holo-RBP concentrations, whereas retinol explained only 14%. Women were classified as vitamin A sufficient (n = 185) or deficient (n = 74) using the RBP:TTR index and were stratified into 3 gestational groups (I: 24-28 wk, II: 29-33 wk, III: >33 wk). Concentrations of free holo-RBP were higher in vitamin A-sufficient women than in vitamin A-deficient women (mean +/- SEM, 48.1 +/- 1.2 vs. 27.6 +/- 0.8 nmol/L; P < 0.001), and in a 3 x 2 factorial analysis, the interaction between gestational group and vitamin A status was significant. These results demonstrate that the RBP:TTR index is a useful proxy for free holo-RBP concentration and that vitamin A status affects its distribution.