Inhibition of deoxyribonuclease I activity was used to assay the actin monomers and the pointed ends of actin filaments in lysates of Dictyostelium discoideum. The KD for the binding reaction was 0.2-0.3 nM. Total cellular actin was 93 microM in monomers (approximately 0.1 fmol/cell) of which roughly half was initially polymeric. Essentially all of the filamentous actin (F-actin) was readily pelleted in the microcentrifuge and was therefore presumed to be in the cytoskeleton. Free F-actin barbed ends, measured as pelletable [3H]cytochalasin B, numbered 1.8 x 10(5)/cell; nuclei for the polymerization of rabbit muscle globular (monomeric) actin numbered 2.0 x 10(5)/cell; and pointed ends, determined by their inhibition of deoxyribonuclease I, numbered 3.6 x 10(5)/cell. These values suggest that half the barbed ends might be occluded. On average, the filaments contained approximately 76 subunits and were therefore about 0.2 micron long. The distribution of their lengths was estimated from the time course of depolymerization following vast dilution. Three populations were defined. In one experiment, the smallest population contained 71% of the F-actin mass and 96% of the pointed ends; these filaments averaged 80 subunits or 0.22 microns in length. An intermediate population contained 14% of the F-actin mass and 3% of the filaments; these were roughly 460 subunits (1.3 microns) long. The largest population contained 15% of the F-actin mass in about 0.3% of the filaments; these were 13 microns in length, about the diameter of the cell. The numerous short filaments might populate a cortical mesh, while the long filaments might constitute endoplasmic bundles.