Blood platelets are particularly rich in cytoskeletal proteins and respond to stimulation and activation by changes in shape. We examined the effect of blood platelet activation on the subcellular distribution of the cytoskeletal proteins, actin, myosin, alpha-actinin and actin-binding protein. These studies were performed with immunofluorescent staining on thin cryosections of paraformaldehyde-fixed platelets and by immunogold labeling of ultrathin cryosections of glutaraldehyde-fixed blood platelets. Platelets were studied immediately at blood collection (resting platelets), in platelet-rich plasma and after gel filtration (partially activated platelets), and after gel filtration and thrombin activation (0.5 U/ml, 10 min, 37 degrees C) (activated platelets). Resting platelets were disk-shaped and showed homogeneous distribution of cytoskeletal proteins. Partially activated platelets were more spherical and showed at least one protrusion. Immunofluorescence and immunogold labeling showed a more intense staining of the peripheral 0.2 to 0.3 micron of cytoplasm of these platelets. In the immunofluorescence photographs this resulted in the appearance of small fluorescent rings with staining at the periphery of cross-sectioned cells. Activated platelets showed an irregular outline composed of broad based pseudopods. Cell centers were composed of poorly delineated electron-dense material, interspersed with profiles of surface-connected tubules. The broad based pseudopods stained uniformely for actin, alpha-actinin and actin-binding protein. The cell center stained poorly for these proteins. Myosin staining was found in the peripheral cortex, but also in the cell center. Partially activated platelets that had returned to the disk shape after incubation at 37 degrees C showed increased submembranous concentration of microfilament proteins. These data reveal the profound cytoskeletal rearrangements that already occur upon minimal platelet activation and emphasize that platelets that have returned to the disk shape are not identical to resting platelets.