Structural proteins of vaccinia virus (IHD-J strain) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. Glycoproteins and phosphoproteins were identified. Correlations between the bands revealed by the continuous buffer method and those developed by the discontinuous buffer method were confirmed by two-dimensional SDS-PAGE, and the spots in the two-dimensional electrophoresis (first, isoelectrofocusing; second, SDS-PAGE) were identified by parallel electrophoresis of selectively solubilized polypeptides. Double labeling of the proteins with [ 3H]fucose and [ 14C]glucosamine showed that the proteins of virus grown in KB cells were glycosylated. When the virus was grown in L929 cells, the carbohydrate moieties lacked fucose, while glucosamine was detected in the same 14 polypeptides as in virus grown in KB cells. All glycopolypeptides migrated as clusters of spots upon two-dimensional electrophoresis. 32P incorporation was found in nine bands, five of which coincided with [ 35Szmethionine-labeled proteins. The phosphorylated polypeptides also migrated as multiple spots upon two-dimensional electrophoresis. The results indicated that the structural polypeptides of vaccinia virus detected as 39 bands in one-dimensional gels and 84 spots in two-dimensional profiles could be assigned to 52 polypeptide species, of which about 50% were basic.