Human leukemia cell lines were examined for the status of the retinoblastoma (RB) protein by immunoblotting analysis using antibodies raised against the TrpE-RB fusion protein. One of 16 cell lines examined, the T-cell acute lymphoblastic leukemia (ALL) line HSB-2, lacked the 110-Kd RB protein. Southern blot analysis of genomic DNA extracted from HSB-2 cells showed a large homozygous deletion of the RB gene, stretching from exon 18 beyond exon 27. Northern blot analysis showed multiple, abnormal RB transcripts in HSB-2. A truncated protein (72 Kd) was detected with 35S-methionine labeling but not with 32P- orthophosphate labeling of the HSB-2 cells. The genomic deletion of greater than 85 kb DNA at the RB locus (13q14) was not detectable in the karyotype of the HSB-2 cells. Among the 16 human leukemia cell lines examined for the status of the RB gene, only one, the HSB-2 line, showed an abnormal RB protein. Further study of primary leukemia and lymphoma samples appears to be warranted.