The mechanism of dominant follicle selection remains obscure. We have investigated the association between follicle diameter and follicular steroid levels in individual human ovarian follicles throughout the menstrual cycle. Fluid from ovarian follicles (n = 326) was obtained in vivo during surgery from 55 regularly cycling women with proven fertility. Follicles were divided into dominant (diameter >9 mm, n = 45) and non-dominant (diameter < or = 9 mm, n = 281) based on ultrasound measurements. Fluid was assayed for oestradiol (E2), androstenedione (AD), and progesterone (P). Median P and E2 levels were significantly lower (P < 0.0001) and AD levels significantly higher (P = 0.03) in non-dominant as compared to dominant follicles. In non-dominant follicles AD (r = 0.14, P = 0.02), but not P and E2, levels were correlated to follicular diameter, and significant changes in steroid concentrations across the menstrual cycle were absent. In dominant follicles, diameter was positively correlated with P and E2 (P < 0.001) levels, and inversely correlated with AD concentrations (P = 0.01). Results indicate that (1) intrafollicular oestradiol concentrations rise only in follicles exceeding 9 mm in diameter and correlate with the diameter of these dominant follicles, suggesting that significant increase in aromatase enzyme activity occurs only in the dominant follicle (2) a cycle-independent accumulation of androstenedione with size occurs in non-dominant follicles, and (3) progesterone production occurs in the largest dominant follicles only, suggesting a limited, if any, role for progesterone during follicle development.