CONTEXT: Limited data suggest that testosterone is decreased during space flight, which could contribute to bone and muscle loss. OBJECTIVE: The main objective was to assess testosterone and hormone status in long- and short-duration space flight and bed rest environments and to determine relationships with other physiological systems, including bone and muscle. DESIGN: Blood and urine samples were collected before, during, and after long-duration space flight. Samples were also collected before and after 12- to 14-d missions and from participants in 30- to 90-d bed rest studies. SETTING: Space flight studies were conducted on the International Space Station and before and after Space Shuttle missions. Bed rest studies were conducted in a clinical research center setting. Data from Skylab missions are also presented. PARTICIPANTS: All of the participants were male, and they included 15 long-duration and nine short-duration mission crew members and 30 bed rest subjects. MAIN OUTCOME MEASURES: Serum total, free, and bioavailable testosterone were measured along with serum and urinary cortisol, serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and SHBG. RESULTS: Total, free, and bioavailable testosterone was not changed during long-duration space flight but were decreased (P < 0.01) on landing day after these flights and after short-duration space flight. There were no changes in other hormones measured. Testosterone concentrations dropped before and soon after bed rest, but bed rest itself had no effect on testosterone. CONCLUSIONS: There was no evidence for decrements in testosterone during long-duration space flight or bed rest.