An association between physical activity and premenopausal breast cancer risk may be due, in part, to relationships with sex hormones or growth factors. Therefore, we assessed whether MET-h/week of total physical activity (moderate-to-vigorous intensity), walking, or vigorous physical activity, and h/week of standing or sitting were associated with plasma concentrations of several hormones. We examined levels of estrogens, androgens, progesterone, prolactin, sex hormone binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), IGF binding protein-3, and growth hormone (GH) in 565 premenopausal women, ages 33-52 years, from the Nurses' Health Study II (NHSII). About 87% of women had both timed follicular and luteal samples; other women had one untimed sample. In general we observed few associations between sex hormone or IGF levels and measures of physical activity or inactivity. However, free testosterone was modestly inversely associated with total physical activity (p-trend = 0.02). Luteal estradiol, free estradiol, and estrone also were inversely associated with total physical activity (p-trend = 0.10, 0.04, 0.01, respectively); however, the trend was substantially attenuated when excluding women with anovulatory cycles or irregular cycles. These cross-sectional results suggest that physical activity and inactivity have limited associations with premenopausal sex hormone and growth factor levels, except possibly luteal estrogens.