The intrinsic tryptophan fluorescence of bacteriophage T4-coded gene 32-protein is found to be partially quenched on binding a variety of mono-, oligo-, and polynucleotides. This phenomenon is exploited to partially "map" the nucleic acid binding site of the protein. The intrinsic fluorescence spectrum of the protein peaks at about 347 nm, compared to 359 nm for the fully solvated model fluorophore, N-acetyl-L-tryptophanamide. Nucleotide binding, or collisional quenching by iodide ion, reduces the intensity of the fluorescence, with little or no peak shift. Small ligands, ranging in size from ribose- and deoxyribose-phosphate to tetranucleotides, quench the fluorescence by 2 to 6%; larger ligands quench from 20 to 35% of the intrinsic protein fluorescence. Iodide quenching experiments subjected to Stern-Vollmer analysis suggest that the binding of short nucleotide-containing ligands brings about a conformational change in the protein, fully exposing a tryptophan side chain to the solvent environment. The fluorescence of this tryptophan is fully quenched by the binding of d(Ap)2, but is largely unaffected by the binding of d(ApA) or d(pA)2, indicating both that this (tryptophan) "reporter" residue is located in the nucleic acid binding site and that binding is polar, i.e. polynucleotide chains of only one orientation are complexed. Long oligonucleotides fully quench the fluorescence of this binding site tryptophan. At high salt concentration (2 M NaCl), gene 32-protein forms self-limited dimers (Carroll, R.B., Neet, K.E., and Goldthwait, D.A. (1972) Proc. Natl, Acad. Sci. U.S.A. 69, 2741-2744; (1975) J. Mol. Biol. 91, 275-291). These dimers, in either high salt or in low salt after cross-linking, fail to bind nucleotides, suggesting that dimer formation partially occludes the nucleic acid binding site and thus that these dimers are probably not involved as intermediates in cooperative protein binding to the DNA. On the other hand, dimerization apparently results in a conformational change which fully exposes the "reporter" tryptophan to iodide quenching. These results are used to formulate a model of some of the nucleic acid-protein and protein-protein interactions involved in the cooperative binding of gene 32-protein to single-stranded DNA.