The nucleotide sequence G-T-Ψ-C-G(A)- has previously been found in every tRNA of known sequence that is active in protein biosynthesis. An exception to this generalization is the recently sequenced initiator tRNA from yeast cytoplasm. It is now reported that cytoplasmic initiator tRNAs from wheat germ, rabbit liver, and sheep mammary gland also lack the G-T-Ψ-C-G(A)- sequence. Thus: ( i ) nucleoside composition analyses show the absence of T in all these tRNAs; ( ii ) analyses of oligonucleotide fragments produced by T1 ribonuclease show the absence not only of the T-Ψ-C-G(A)- sequence, but also of U-Ψ-C-G(A)- or U-U-C-G(A)- sequences in such digests. The absence of G-T-Ψ-C-G(A)- in the eukaryotic cytoplasmic initator tRNAs is, therefore, not simply due to lack of enzymatic modification of U to T.