The p53 is a nuclear protein that is associated with normal cellular proliferation and can cooperate with Ha-ras in causing cellular transformation in vitro. Lineage association is known to exist between p53 expression and normal lymphopoiesis, but not myelopoiesis. We studied the expression of p53 using chronic myelogenous leukemia (CML) cell lines, somatic hybrids of these cells, and leukemic cells from CML patients. Lymphoid CML lines expressed both p53 mRNA and protein. We also analyzed p53 synthesis by two B-lymphoid lines from the same CML patient; cells of one line were derived from the neoplastic clone, cells of the other were derived from the normal clone. Both synthesized equal amounts of a phosphorylated p53 protein. None of the myeloid CML lines expressed detectable p53 protein and two of four expressed negligible p53 mRNA. Two other myeloid CML lines and myeloid cells from three of four patients expressed p53 mRNA. These findings suggest that expression of the gene is not regulated normally in CML. Several approaches were pursued to explore the differential expression of p53. Southern blot analyses showed no gross alterations in the p53 gene from cells of either the expressing or the nonexpressing lines. No difference in the pattern of demethylated CpG sites was noted in the region of the p53 gene in cells from K562 (myeloid p53 nonexpressor) and in BV173 (lymphoid p53 expressor). The sites of demethylation clustered in and around the p53 promoter in both cell lines. Somatic hybrids formed between a p53 mRNA nonexpressor myeloid line (K562) and the parental p53 expressor lymphoid lines (Daudi, PUT) produced p53 mRNA and protein, suggesting that p53 is a dominantly expressed protein and that lack of expression in myeloid cells is not mediated by a trans-acting negative regulatory protein. DNA transfection experiments performed using the indicator gene chloramphenicol acetyltransferase attached to promoter sequences of p53 showed that these constructs were equally activated in BV173 (p53 expressor) and K562 (p53 mRNA nonexpressor). The mechanism of p53 regulation in CML remains unclear.