Three α-tubulins and two β-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for β-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The α1- and β1-tubulins were detected in both myxamoebae and plasmodia; α2 and β2 were detected only in plasmodia, α3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the α1-, α2-, β1-, and β2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.