Phagosomes with pathogenic mycobacteria retain fusion and intermingling characteristics of early endosomes indefinitely. The time course of acquisition of newly endocytosed tracers becomes, however, atypical (lag instead of immediate acquisition) starting from day 1 postinfection (p.i.), thereby suggesting that additional factors affect this process. Disruption of the actin filament (F-actin) network by cytochalasin D perturbs the movement of early endosomes and probably fusion events among early endosomes and phagosomes. Here we compare, by immunofluorescence microscopy, the morphology and distribution of F-actin in macrophages infected with virulent Mycobacterium avium, in uninfected macrophages, or in macrophages after phagocytosis of nonpathogenic bacteria (Mycobacterium smegmatis or Bacillus subtilis) or hydrophobic latex particles. In uninfected cells, F-actin appeared as a network of small filaments distributed throughout the cell; about 80% of the cells also displayed one or two small patches of F-actin at the cell periphery. Virulent M. avium caused a marked disorganization of the F-actin network starting from day 1 p.i. The most salient features were the formation of several large patches, the progressive disappearance of the small filaments, and the appearance of large numbers of tiny punctate structures starting from day 2 p.i. With the three other particles, the F-actin network was unmodified compared to that in uninfected cells. The atypical lag in acquisition of newly endocytosed tracers by M. avium-containing phagosomes, therefore, seems to coincide with the disorganization of the F-actin network.