Abstract Context: Associations between menstrual cycle length and chronic diseases are hypothesized to be due to differences in underlying hormonal patterns. Objective: The aim of the study was to evaluate the association between menstrual cycle length and the hormonal profile and anovulation. Design and Setting: We conducted a prospective cohort study at the University at Buffalo from 2005 to 2007. Participants: We recruited 259 healthy, regularly menstruating women aged 18–44 yr. Main Outcome Measures: Cycle length was observed for up to two cycles. Serum estradiol, progesterone, LH, and FSH were measured up to eight times per cycle for up to two cycles. Results: Women with short cycles (<26 d) had higher FSH concentrations during menses and in the late luteal phase, higher follicular estradiol concentrations, and lower LH concentrations across the cycle. Among women with longer cycles (>35 d), estradiol and LH peaks occurred on average about 3 d later, and FSH peaks about 1 d later compared to women with normal-length cycles. Both short and long cycles, compared with normal-length cycles, had an increased probability of anovulation. In general, per-cycle exposure to hormones was less in short cycles based on the area under the curve, although over time the cumulative exposure to estradiol would be greater for women with short cycles. Conclusions: Short ovulatory cycles were associated with higher follicular phase estradiol, an earlier rise in FSH, and an increased risk of anovulation. These results suggest that menstrual cycle length may be a relevant indicator of estradiol exposure and risk of anovulation among regularly cycling women.