Major cytoplasmic GTP-binding proteins in Dictyostelium discoideum were identified by direct photoaffinity labeling with [α- 3 2 P]GTP. Three actin-binding proteins and a protein with an apparent molecular mass of 24 kDa (p24) could be labeled with [α- 3 2 P]GTP. p24 binds to DEAE-cellulose, behaves like a monomer during gel filtration and was purified to homogeneity by GTP-affinity chromatography. In comparison to other nucleotide triphosphates the binding of GTP to p24 is highly specific.