Walker 256 rat carcinosarcoma cell lines sensitive (WS) or resistant (WR) to bifunctional nitrogen mustards have modal chromosome numbers of 60 and 55 respectively. Karyotype analysis revealed that these cell lines have retained the major marker chromosomes present in the original in vivo Walker tumours. One new marker chromosome, a metacentric, was found in the WR cell line. C-banding revealed that in the WS cell line the secondary constriction of the marker chromosome was stained, whereas no staining was found on this chromosome in the WR cell line. Three autosomes containing very prominent non-centromeric C-bands were present in WS but not in the WR cell line which has 2 other chromosomes with minor C-bands. As non-centromeric C-bands do not occur in the normal rat karyotype, these are easily identifiable specific tumour markers for these two cell lines.