I demonstrate that the U1 snRNAs of human cells are heterogeneous in sequence. Polyacrylamide gel and RNase T1 fingerprint analyses of U1 RNAs isolated from a variety of human cultured cells, including HeLa, 293, K562 and NT2/D1, show that minor variants of the human U1 RNA (hUla) comprise between 5% and 15% of the total U1 RNAs in these established cell lines. The patterns of variants are cell line specific, suggesting that expression of these minor species of hUla RNAs reflect polymorphisms of the hUla true genes rather than existence of an additional class of human embryonic U1 genes. Also, the hUla variants described here are not the products of previously identified human U1 Class I pseudogenes.