STUDY QUESTION Do ovulatory hormone profiles among healthy premenopausal women differ between women with and without sporadic anovulation? SUMMARY ANSWER Women with one anovulatory cycle tended to have lower estradiol, progesterone and LH peak levels during their ovulatory cycle. WHAT IS KNOWN ALREADY Anovulation occurs sporadically in healthy premenopausal women, but the influence of hormones in a preceding cycle and the impact on a subsequent cycle's hormone levels is unknown. STUDY DESIGN, SIZE, DURATION The BioCycle Study was a prospective cohort including 250 healthy regularly menstruating women, 18–44 years of age, from Western New York with no history of menstrual or ovulation disorders. The women were followed with up to eight study visits per cycle for two cycles, most of which were consecutive. PARTICIPANTS/MATERIALS, SETTING AND METHODS All study visits were timed to menstrual cycle phase using fertility monitors and located at the University at Buffalo women's health research center from 2005 to 2007. The main outcomes measured were estradiol, progesterone, LH and follicle-stimulating hormone levels in serum at up to 16 visits over two cycles. Anovulation was defined as peak serum progesterone concentrations ≤5 ng/ml and no serum LH peak detected during the mid- or late-luteal phase visit. MAIN RESULTS AND THE ROLE OF CHANCE Reproductive hormone concentrations were lower during anovulatory cycles, but significant reductions were also observed in estradiol (−25%, P = 0.003) and progesterone (−22%, P = 0.001) during the ovulatory cycles of women with one anovulatory cycle compared with women with two ovulatory cycles. LH peak concentrations were decreased in the ovulatory cycle of women with an anovulatory cycle (significant amplitude effect, P = 0.004; geometric mean levels 38% lower, P < 0.05). LIMITATIONS, REASONS FOR CAUTION Follow-up was limited to two menstrual cycles, and no ultrasound assessment of ovulation was available. Data were missing for a total of 168 of a possible 4072 cycle visits (4.1%), though all women had at least five visits per cycle (94% had seven or more per cycle). WIDER IMPLICATIONS OF THE FINDINGS These results suggest a possible underlying cause of anovulation, such as a longer-term subclinical follicular, ovarian or hypothalamic/pituitary dysfunction, even among healthy, regularly menstruating women. STUDY FUNDING/COMPETING INTERESTS This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD (Contract no. HHSN275200403394C). The authors have no potential competing interests.