Polar organic solvents, such as N-methylformamide (NMF), N,N-dimethylformamide, and dimethyl sulfoxide, have been demonstrated to induce differentiation in a number of neoplastic cell lines, including human colon cancer cells. Although the mecha nism of action of these agents is yet unknown, one possibility is that polar solvents induce a change in lateral mobility of mem brane lipids, important to the maturational process. To determine the relationship between polar solvent treatment and changes in membranes, we examined the effects of exposure to NMF on membrane fluidity in human colon cancer cells (DLD-1; clone A). Membrane viscosity was assessed by determining lipid lateral diffusion following photobleaching of a fluorescent lipid probe in individual intact cells. Exposure of cells to NMF led to a significant increase in membrane viscosity following 2 days of treatment, with maximal changes occurring after 11 days. NMF induced these effects over a limited concentration range with 1.0% NMF in the medium having the maximal effect, and 0.5% or 1.5% having less or no effect. Growth of cells with N,N-dimethylformamide (0.8%) also led to increases in membrane viscosity. The observed membrane changes correlated well with the effect of NMF on differentiation in these cells as previously reported, as well as with cell growth rate and morphology in the present study. The increase in viscosity caused by prolonged NMF treatment was reversible, with a return to untreated levels by 9–11 days after removal of NMF. Thus, there is a strong correlation between the attainment of more benign, better differentiated phenotype in polar solvent-treated clone A cells and increases in membrane viscosity.