The distribution of actin in proteose peptone-elicited murine peritoneal macrophages is examined with fluorescent analog cytochemistry (FAC), immunofluorescence, and electron microscopy (EM). Living adherent macrophages, microinjected with 5-iodoacetamidofluorescein-labeled actin, show a rather uniform distribution of actin with punctate and linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of the microinjected actin into the cell's actin cytoskeleton is also demonstrated when microinjected cells are subsequently examined for fluorescein fluorescence after fixation and extraction. However, a substantial perinuclear pool of actin, observed with FAC, is lost when microinjected cells are prepared for immunofluorescence using standard fixation methods. These results suggest that part of the cellular actin, possibly nonfilamentous or oligomeric, can be extracted during the normal preparative steps for immunofluorescence. When the dynamic distribution of actin structures is examined in living cells, extension of the cell's periphery is associated with the formation of punctate structures. The distribution of the most stable, nonextractable actin structures in fixed cells at different stages of spreading is quantified using rhodamine-labeled phalloidin and antiactin indirect immunofluorescence. At early stages, the rounded cells show cortical bands of fluorescence surrounding the nuclear region with punctate structures directly above the plane of the attached plasma membrane. At later time periods, fully spread cells contain both punctate and linear fluorescent structures. Adherent macrophage membranes, a preparation in which the attached membrane and membrane-cortex are isolated by shearing away the unattached plasma membrane and underlying cytoplasm, show punctate and linear fluorescence when stained with rhodamine-labeled phalloidin. When the same cell remnant is negatively stained and examined with EM, the fluorescent punctate structures coincide with electron-dense foci and associated radiating thin filaments. We suggest that the optimal approach for elucidating the distribution of cytoskeletal and contractile proteins involved in motile processes is a combined approach using all three techniques. Although each technique is subject to potential artifacts and limitations, the use of FAC can permit the visualization of both the soluble and stabilized components of the cytoskeleton in living, functional cells. A qualitative method for determining differences in local concentrations of proteins is also presented.