Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[35S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (alpha 1, alpha 2, beta 1, and beta 2) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to alpha-tubulin. Only trace amounts of alpha- and beta-tubulin and action were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and alpha- and beta-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of alpha- and beta-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to alpha-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.