T lymphocytic colony formation by peripheral lymphocytes separated by discontinuous albumin gradient centrifugation was evaluated in 8 patients with Philadelphia (Ph1-positive chronic myeloid leukemia (CML). Colonies were obtained using a liquid-on-agar culture system recently introduced (PHA overlayer-leukocyte feeder layer assay) which has been shown to be simple and reliable. The pattern of colony growth in CML and in normal controls was similar, the peak ranging from the 4th to the 6th day. Also the morphological aspects of colonies did not differ in the two groups. Cells recovered from CML lymphocytic colonies were shown to belong to T cell lineage, as they are able to form spontaneous E-rosettes and to respond to mitogenic stimulation in vitro. In contrast, cells recovered from all other cultured fractions failed to display these properties.Cytogenetic analysis showed that T colony cells were Ph1-negative whereas the chromosome anomaly was found in nonlymphoid colonies of the same patients, thus suggesting a nonclonal origin of T lymphocytes in CML.