A cDNA clone for a basic putative actin microfilament-associated protein, p27, highly induced in serum-stimulated NIH 3T3 cells, has been isolated by polyclonal antibodies and sequenced. p27 mRNA is a 1.2-kb molecule which is very low in resting NIH 3T3 cells but can be induced at least 100 times after 8 h of fetal calf serum stimulation. In contrast to other inducible mRNAs, p27 mRNA is stable, and its levels can be superinduced by cycloheximide mainly by prolonging transcription. The lack of expression of this messenger in mouse tissues, as well as in all cell lines so far tested, suggests that p27 may be an fibroblast-specific protein. One major open reading frame found in p27 cDNA codes for a 201 amino acid polypeptide not related to any previously described actin-binding protein. Interestingly, it shows alternative hydrophilic and hydrophobic domains of amino acids symmetrically arranged from the middle of the protein. The coordinate induction of p27 and actin mRNAs suggest that p27 may be involved in the cytoskeletal rearrangements induced early in cell growth and proliferation.