The transfer RNAs for aspartic acid, asparagine, histidine, and tyrosine respond to codons in the third column of the genetic code and contain a hypermodified nucleoside known as queuosine (Q) in the first position of the anticodon of the major isoacceptor tRNA. Nothing is known about the physiological or biochemical function of Q. Germ-free mice were maintained for a period of nine tRNA half-lives on a chemically defined diet known to contain all essential constituents of the rodent diet but no Q or its base, queuine. The tRNAs for histidine and asparagine contained only 15% of the Q-containing isoacceptor tRNA. On the other hand, the Q-containing isoacceptor comprised 88% of the tRNAHis and 85% of the tRNAAsn in conventional mice and germ-free mice fed commercial mouse chow. Transfer RNAAsp and tRNATyr were completely modified with respect to Q in germ-free mice maintained on the chemically defined diet as well as on normal mouse chow. Germ-free mice fed the chemically defined diet contained normal amounts of the hypermodified base wye in tRNAPhe.