We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.