The position of an alternative polyadenylation [poly(A)] site at the 3' end of the polyomavirus middle T antigen (T-Ag) coding sequences suggests the possibility of a functional role for this site in early gene regulation. The fine structure of this alternative poly(A) site was determined by cDNA sequence and 3' S1 analyses. Cleavage-poly(A) was found to be heterogeneous, occurring at multiple CA dinucleotides downstream from the AATAAA signal sequence. About 50% of the alternative poly(A) takes place upstream from the middle T-Ag stop codon. In addition, the pattern of splicing of transcripts with the alternative poly(A) site differed from that with the major poly(A) site at the end of the early region. The ratio of the small and middle T-Ag splices to the large T-Ag splice for the alternative poly(A)+ mRNAs is about 2.5 times that found for mRNAs with the major poly(A) site. The altered splicing pattern and 3'-end heterogeneity of the alternative poly(A)+ mRNAs would result in preferential translation of small T-Ag (to a greater degree) and middle T-Ag over large T-Ag at later times in the polyomavirus lytic cycle.