OBJECTIVE Our purpose was to investigate possible relationships with spectrophotometric absorbance (458-nm region) and biochemical variables in follicular fluid (FF) as well as in vitro fertilization (IVF) outcome. METHODS This study included 227 normal ovulatory women undergoing oocyte retrieval for IVF. Blood-uncontaminated fluid samples, identified by spectrophotometry, were investigated. Spectrophotometric absorbance of FF at 458 nm (n = 426), as well as hLH, FSH, PRL, hCG, testosterone, sialic acid, alpha 1-antitrypsin and plasminogen of selected fluids, was analyzed. RESULTS Small-volume follicles (< or = 2 ml) were associated with higher absorbance profiles (P < 0.05), when compared to volumes greater than 2 ml. Our data suggest that the presence or absence of an oocyte, the potential of an oocyte to fertilize or cleave, failed to show any relationship with maximum FF absorbance at 458 nm. Maximum absorbances were significantly lower in FF from patients who subsequently became clinically pregnant (P = 0.039). No correlation between FF absorbances and biochemical parameters (P > 0.15) were established. CONCLUSIONS Absorbance of clear FF at 458 nm should not be viewed as the single parameter to predict oocyte development in vitro.