To better understand the changes that occur in cytoplasmic actin during cell movement, we studied the effect of inhibitors of cell movement on the molecular conformation of actin and its incorporation into the Triton-insoluble cytoskeleton of human neutrophils. The sulfhydryl reactive compound N-ethylmaleimide caused an increase in cellular F-actin as measured by uptake of the F-actin specific fluorescent probe 7-nitrobenz-2-oxadiazole-phallacidin. However, N-ethylmaleimide reduced the amount of actin associated with the Triton-insoluble cytoskeleton. Dithiobisnitrobenzoic acid, a sulfhydryl reagent that does not cross cell membranes efficiently, did not alter the F-actin content of neutrophils. The effect of N-ethylmaleimide was blocked by the presence of dithiothreitol, a donor of sulfhydryl groups. N-ethylmaleimide did not affect the polymerization of actin in a cell-free system. Cytochalasin B did not alter F-actin content of neutrophils but did decrease actin in cytoskeletons of resting neutrophils. Cytochalasin inhibited the increase in F-actin initiated by the chemoattractant N-formylmethionylleucylphenylalanine. We propose that N-ethylmaleimide blocks the stabilization of G-actin in cytoplasm, interferes with the incorporation of F-actin polymer into the cytoskeleton, and depolymerizes the cytoskeleton. In contrast cytochalasin stabilizes G-actin in the presence of chemotactic peptide. These data suggest that reversible conversion of G-actin to F-actin and incorporation of F-actin into the Triton-insoluble cytoskeleton are important for neutrophil movement.