Cytochalasin B competitively inhibits the transport of 2-deoxy-D-glucose and thymidine in a number of different cell lines (Novikoff rat hepatoma cells, mouse L, S180 and Ki-MSV-transformed BALB/3T3 cells, and human HeLa cells). The apparent Km values for the transport of these substrates as well as the apparent Ki values for the inhibition by cytochalasin B are very similar for the various cell lines, and the effect is readily and completely reversed by removal of the chemical. Thymidine transport by Chinese hamster ovary cells however, is little affected by cytochalasin B, whereas the transport of 2-deoxy-D-glucose, uridine and guanine by these cells is competitively inhibited to about the same extent as in other cell lines. In addition and concomitant with the inhibition of cytokinesis and an alteration in cell shape, cytochalasin B also impairs and delays the formation of functional transport sites for thymidine, guanine and choline in synchronized populations of Novikoff cells without affecting the apparent affinities of the transport systems for their substrates. This effect is unrelated to the direct inhibition of the transport processes, since the drug does not directly inhibit choline transport and has no effect on the formation of 2-deoxy-D-glucose transport sites in spite of the fact that it strongly inhibits the transport of this substrate. The inhibition of functional transport sites may be due to the induction of a structural alteration in the membrane by cytochalasin B which impairs the insertion of new proteins of certain but not all transport systems into the membrane.