Leukemia inhibitory factor/D-factor, a potent differentiation-inducing glycoprotein for murine myelomonocytic leukemic M1 cells, rapidly stimulated the phosphorylation of a 27 kDa protein with an isoelectric point of 5.6 in a LIF-sensitive M1-T22 cell line but not in a LIF-resistant M1-D(-) cell line. The increase in phosphorylation was detectable 5 min after LIF treatment and was maximal at 10 min. Heat shock treatment at 44.5 degrees C for 30 min also induced the phosphorylation of the same 27 kDa protein. Although this 27 kDa protein did not become labeled with [35S]-methionine, metabolic labeling experiments using [35S]-cysteine or [3H]-leucine clearly demonstrated that the synthesis of this protein was enhanced after heat shock. These results suggest that the phosphorylated 27 kDa protein is a low molecular weight stress protein and that the protein may play a role at an early stage in the LIF signaling pathway probably linked to macrophagic differentiation.