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In order to investigate the 5' terminal structural heterogeneity of the 16S size class of SV40 late RNA, we have bound an SV40 DNA fragment labeled at its 5' termini with P32 to the .939-.945 map unit region of late lytic cytoplasmic polyadenylated RNA, used reverse transcriptase to prepare cDNA copies of the 5' termini of this RNA, separated the cDNA products on an 8% polyacrylamide-7 M urea gel and subjected these products to nucleic acid sequence analysis. A number of discrete cDNAs were obtained. Analysis of these cDNAs has suggested the presence of three categories of 16S species all containing the same body extending from residues 1381-2592 (.939-.170 m.u.) but differring in the structure of their leader segments. Members of the first category contain leaders which are colinear with SV40 DNA, have a common 3' terminus at residue 444 and extend varying distances in a 5' direction. The most abundant 16S species contains a leader of 203 nucleotides and is a member of this group. RNAs of the second category contain leaders with an internal gap between residues 211-352. The single RNA comprising the third category contains a leader with a tandem repetition of nucleotides 351-443 at the 3' terminus of its leader.