The nucleotide sequences at the 5' ends of polyoma virus late mRNA's were determined by direct RNA sequencing of decapped and 5'-(32)P-labeled RNase T1 oligoribonucleotides. Virus-specific polyadenylated cytoplasmic RNA, which was isolated from mouse cells late during productive infection, was enzymatically or chemically treated to specifically remove the cap structure (m(7)Gppp). The unblocked 5' ends of the viral mRNA's were then labeled enzymatically with (32)P, and the RNAs were digested with RNase T1 and fingerprinted. Three oligonucleotides derived from capped termini were isolated, and their sequences were determined to be pGmACAUUUUCUAUUUUAAGp, p(m)AmCAUUUUCUAUUUUAAGp, and p(m)AmUUUUCUAUUUUAAGp. These oligonucleotides comprise a staggered set with members 15, 17, and 18 nucleotides long, which share a common 3' sequence for 15 nucleotides. The sequences correspond exactly to the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606-618, 1980) from 66.79 to 66.46 map units (between 75 and 92 nucleotides preceding the ATG initiation codon for the capsid protein VP2). Previous results showed that the sequence between 13 and 64 nucleotides preceding the VP2 initiation codon corresponds to oligonucleotides reiterated in the leader sequence which is spliced onto the bodies of the three functionally distinct viral late mRNA's (Flavell et al., Cell 16:357-372, 1979; Legon et al., Cell 16:373-388, 1979). The three capped oligonucleotides we sequenced are derived from the first large predicted T1 oligonucleotide 5' to those detected in the leader sequence. The occurrence of a cap at each purine of a single tetranucleotide sequence reflects micro-heterogeneity either in transcriptional initiation or in processing cleavage involved in cap syntheses.