Sublethal doses of 5-FU inhibit division of O-2A/OPCs. Clonal analysis was used to study the effects of low-dose 5-FU (0.05 μM for 24 h) on the division and differentiation of freshly isolated progenitor cells. O-2A/OPCs were grown at clonal density and exposed one day after plating to (a) vehicle alone or (b) 0.05 μM 5-FU for 24 h, doses that killed less than 5% of O-2A/OPCs in mass culture. The number of undifferentiated O-2A/OPCs and differentiated cells (oligodendrocytes) was determined in each individual clone from a total number of 100 clones in each condition by morphological examination and by immunostaining with A2B5 and anti-GalC antibodies (to label O-2A/OPCs and oligodendrocytes, respectively). Results are presented as three-dimensional graphs. The number of progenitors per clone is shown on the x (horizontal) axis, the number of oligodendrocytes on the z (orthogonal) axis and the number of clones with any particular composition on the y (vertical) axis. In 5-FU-treated cultures analyzed five days after initiating 5-FU exposure, there was an increase in the representation of small clones consisting wholly of oligodendrocytes and clones containing large numbers of oligodendrocytes, a reduction in the representation of large clones, a general shift of clone size towards smaller values, and a clear reduction in the total number of progenitor cells (see text for details). Experiments were performed in triplicate in at least two independent experiments.