The levels of thiamine, riboflavin, and nicotinic acid in the rumen of sheep, fed a variety of diets, were determined by chemical methods. The intakes of dry matter, B vitamins, protein, non-protein nitrogen, and energy were recorded, and the rumen volumes on each diet were estimated. The diets were mainly of a semi-synthetic type, consisting of sodium hydroxide-treated straw supplemented with varying amounts of casein, urea, starch, sugar, and minerals. When the intake of the B vitamins was high, little microbial synthesis took place in the rumen. However, when the diet offered was deficient in the R vitamins the levels of synthesis were high. There was no apparent relationship between the levels of the vitamins in the food and the levels in the rumen liquid. However, a relationship between the nitrogen intake and the vitamin levels in the rumen was found. This relationship was marked when widely differing levels of B vitamin were fed. The total substitution of the dietary protein (casein) by non-protein nitrogen (urea) did not influence the synthesis of thiamine and riboflavin but did depress the synthesis of nicotinic acid.