The U1 RNA species in 10 mouse cell lines were examined by two-dimensional polyacrylamide gel electrophoresis. Seven cell lines that were not infected by Friend spleen focus forming virus gave only one (I) or two (I and II) U1 RNA-containing spots. However, two Friend cell lines (FVTCT and Friend 745a cells) gave three spots (I, II, and III) and another Friend cell line, K-1 cells, gave four spots (I, II, III, and IV). As a result of further separation and fingerprinting analysis of each spot, FVTCT and Friend 745a cells were found to contain U1a-1, U1b-1, -2, and -6 RNAs whereas K-1 cells were found to contain several U1 RNAs, which we call U1a-1 and -2, U1b-4, -5, and -6 RNAs. We determined the sequences of these seven U1 RNAs and found that mouse U1 RNAs had two basic sequences (U1a and -b). The nucleotide sequence of U1a-1 RNA was identical to that of rat U1a RNA, while U1a-2 RNA was one base different from U1a-1 RNA. Relative to U1a-1 RNA all of the U1b RNAs had five base substitutions and one additional base and were under-methylated in the center. U1b-6 RNA contained two base substitutions and one base addition in the 3'-terminal portion of U1b-1 RNA. U1b-2, -4, and -5 RNAs, which were observed only in Friend cells, each had an additional base substitution in the 5'-half of U1b-1 RNA.