Groups of four germ-free (GF) and conventional (CV) rats were given purified diets containing either 50 or 200 g lactalbumin/kg for 2 wk and their urinary excretion of nitrate was measured. Urinary excretion of N-nitrosoproline was also measured in one of the three experiments. Both GF and CV rats given the high-protein diet excreted significantly more nitrate and N-nitrosoproline than those given the low-protein diet. On both diets GF rats excreted more nitrate than their CV counterparts but N-nitrosoproline excretion was not affected by environment. Groups of 11 GF and CV rats given diets containing sesame meal with or without a supplement of lysine-HCl for 2 wk, excreted similar amounts of nitrate on both diets, but more nitrate was excreted by GF rats than by their CV counterparts. N-nitrosoproline excretion by rats given the lysine supplement was higher in both environments. It is concluded that endogenous synthesis of nitrate is mediated by mammalian tissues rather than microflora and that dietary protein is an important source of nitrogen for the synthesis, although surplus amino acids from an imbalanced protein source do not act as precursors of endogenously formed nitrate. Some of the synthesized nitrate or its precursors appears to be metabolized by the microflora in the CV rat.