Incubation of stripped rough microsomes (SRM) with the catalytic subunit of protein kinase A (PKA) permitted specific phosphorylation of seven proteins having relative molecular mass values of 55, 35, 23, 22.5, 22, 18.5 and 16.5 kDa (P55, P35 etc.). By two dimensional gel analysis, we compared these phosphoproteins with low-molecular-weight GTP-binding proteins and revealed that P23 and P22.5 co-migrated with known GTP-binding proteins. Next we examined the effect of cAMP-dependent phosphorylation on a GTP-dependent membrane function, membrane fusion. Quantitative analysis indicated no difference in the amount of membrane fusion obtained whether SRM were incubated in the absence or in the presence of PKA. Thus several rough microsomal proteins underwent cAMP-dependent phosphorylation and this post-translational modification did not affect GTP-dependent membrane fusion in a cell free system.