In order to clarify the effects of fish meal supplementation and environmental temperature on nitrogen excretion in feces and urine, nitrogen balance trials were conducted by L 16 table of orthogonal array with 8 Holstein lactating cows in environmental controlled rooms (18°C 60% relative humidity (RH), 28°C 40% RH and 28°C 80% RH). The results obtained were as follows; 1) there was no difference in fecal nitrogen excretion by fish meal supplementation. However, urinary nitrogen excretion decreased significantly and milk nitrogen and retained nitrogen tended to increase. 2) Nitrogen intake in 28°C was less than in 18°C, but urinary nitrogen increased and the efficiency of nitrogen utilization decreased as temperature increased.