The purpose of this study was to clarify the effects of dietary protein levels on protein nutritional status in rats kept under constant darkness. Thirty-six 4-wk-old female rats (F344 strain) were divided into six groups. Each group was given a diet with one of three different protein levels and kept under normal light and dark cycles (7:00-19:00 light period/19:00-7:00 dark period, N group) or under constant darkness (D group) for 4 wk. The protein levels of the diets were 10%, 20%, and 30% casein. The six groups are referred to as the N10%, N20%, N30%, D10%, D20%, and D30% groups. Body weight gain was low in the D groups, and that in the D30% group was much lower than that in the N30% group. The D30% group retained less nitrogen than the N30% group. As for the amount of urinary nitrogen excreted every 4 h, the values for the D-groups were higher than those for the N-groups in the 11:00-15:00 periods, and that for the D30% group was higher than that for the N30% group in the 15:00-19:00 periods, which means that protein catabolism was higher in the D30% group. It was shown that when rats kept under constant darkness were fed a high-protein diet for 4 wk, their nitrogen retention decreased and their protein nutritional state dropped.