The preparation of fluorescent profilin analogs for binding and spectroscopic studies, in vitro and in vivo, has been hampered by the poor chemical reactivity of this protein in its native form. We have addressed this problem by labeling a mutant, chemically reactive form of profilin. Site-directed mutagenesis was first used to replace a serine residue in a non-essential domain with a reactive cysteine residue. The mutant protein was expressed in Escherichia coli and reacted with tetramethylrhodamine iodoacetamide. In vitro assays indicated that the fluorescent profilin maintained its ability to bind actin, polyproline, and PIP2, to inhibit actin polymerization, and to stimulate actin nucleotide exchange. Fluorescence spectroscopy showed that neither the excitation nor the emission of the analog was sensitive to the interaction with actin or polyproline. However, binding of PIP2 caused a 75% quenching of the fluorescent signal, suggesting a dramatic change in the immediate environment of the probe. When the fluorescent profilin was microinjected into living NRK cells, it became localized at cell-cell junctions and discrete sites near the anterior end, where it colocalized with aggregates of unpolymerized actin. Different engineered forms of profilin with fluorophores located at defined sites should greatly facilitate the study of its interactions with various ligands and cellular structures.