The polyoma mRNAs present in the cytoplasm of primary cultures of mouse kidney cells during lytic infection were characterized by sedimentation velocity analysis and by hybridization to polyoma DNA fragments generated by a specific endonuclease of Hemophilus parainfluenzae (Hpa II). Three distinct species of polyoma mRNAs were found and mapped on the viral genome: (a) early 19S polyoma mRNA, which is complementary to about 45% of the early strand and maps from approximately 78 (5' end) to 23 (3' end) map units; and (b) two related late mRNAs, a larger species (19 S), which is complementary to about 44% of the ate strand and extends from approximately 70 (5' end) to 26 (3' end) map units, and a smaller species (16 S), which is complementary to about 31% and maps between 61 (5' end) and 30 (3' end) map units. Thus, the entire sequence of the late 16S mRNA is comprised within the late 19S mRNA, a finding which was also supported by two-dimensional chromatographic analysis of the RNase T1 oligonucleotides of the two late mRNAs.