Context. To our knowledge, no one has compared the prepractice hydration status of male and female National Collegiate Athletic Association (NCAA) Division I athletes or has studied the effects of the menstrual cycle phase on women's prepractice hydration status. Objective: To report prepractice hydration status of collegiate athletes and determine the factors that might influence that status. Design: Cross-sectional, descriptive study. Setting: University sports team practices. Patients or Other Participants: Participants included 138 male and 125 female athletes (age 19.9 +/- 1.3 years, height = 165.8 +/- 42.9 cm, mass = 77.4 +/- 17.5 kg) from an NCAA Division I New England university. Intervention(s): One spontaneously voided (spot) urine sample was collected from each participant before his or her team practice and was measured 2 times. Main Outcome Measure(s): A refractometer was used to analyze the amount of light that passed through a small drop of urine and assess urine specific gravity. Fluid intake and menstrual history for women were also collected. Three hydration-status groups were defined based on the American College of Sports Medicine and National Athletic Trainers' Association criteria: (1) euhydrated, which was urine specific gravity less than 1.020; (2) hypohydrated, from 1.020 to 1.029; and (3) significantly hypohydrated, equal to or more than 1.030. Results: Thirteen percent of student-athletes appeared significantly hypohydrated, with a mean urine specific gravity of 1.031 +/- 0.002 (chi(2) = 12.12, P < .05); 53% appeared hypohydrated, with a mean urine specific gravity of 1.024 +/- 0.003 (chi(2) = 12.12, P < .05); and 34% appeared euhydrated, with a mean urine specific gravity of 1.012 +/- 0.005 (chi(2) = 0.03, P > .05). A greater percentage of men (47%) than women (28%) were hypohydrated (chi(2) = 8.33, P < .05). In women, no difference was evident between the luteal and follicular phases of their menstrual cycles (chi(2) = 0.02, P > .05). Conclusions: Before activity, athletes were hypohydrated at different levels. A greater percentage of men than women were hypohydrated. Menstrual cycle phase did not appear to affect hydration in women.