A direct competitive heterologous enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine follicular fluid estradiol levels of different antral follicular sizes (small, medium, and large) obtained from the ovaries of heifers in the follicular phase of the estrous cycle. Polyclonal antiestradiol antibodies were raised in New Zealand White rabbits using 6-keto-1,3,5(10)-estratriene-3,17 beta-diol 6-carboxymethyloxime: bovine serum albumin as immunogen and characterized by the usual methods. Horseradish peroxidase conjugated to 1,3,5(10)-estratriene-3,17 beta-diol 3-hemisuccinate was used as a label. The concentration range used for the standard curve was 0 to 1 ng per well. The low detection limit of the technique was 0.3 pg per well with a sensitivity at 50% binding of 93.62 pg per well. Intraassay and interassay CV (%) were < 5.3% and < 7.0%, respectively (n = 10). The recovery rate of the known estradiol concentrations added to follicular fluid averaged 96.1 +/- 1.3%. Compared with a radioimmunoassay (RIA), the values of ELISA were highly correlated (r = 0.99, P < 0.005). This assay was used to quantify follicular fluid estradiol concentrations without previous extraction in three antral follicular sizes; small (< 5 mm), medium (5.1 to 10 mm), and large (10.1 to 20 mm). The mean +/- SE follicular fluid estradiol concentrations were 77 +/- 5.2 ng/ml (n = 490) in small follicles, 111 +/- 19 ng/ml (n = 65) in medium follicles, and 496 +/- 144 ng/ml (n = 45) in large follicles.(ABSTRACT TRUNCATED AT 250 WORDS)