Equilibrium and kinetic studies of the interaction of gene 32 protein of T4 phage with single-stranded fd DNA were performed monitoring the changes in protein fluorescence. From the fluorescence titrations, it was estimated that a monomer of gene 32 protein covered six nucleotide bases on the DNA and the lower limit for the apparent association constant was 1.9 × 108 M-1 with a cooperative parameter of 103 in 0.1 M 2-amino-2-hydroxymethyl-l,3-propanediol hydrochloride (pH 7) at 25 ΰC. When an ionic strength jump was applied to the gene 32 protein-fd DNA complex using a stopped-flow apparatus, the complex underwent a dissociation into its individual components accompanied by an increase in protein fluorescence. The kinetics of the dissociation are not consistent with a single first-order process. The data, however, can be analyzed in terms of a model in which gene 32 protein molecules release cooperatively starting from either one or both ends of a cluster of proteins bound to fd DNA. This type of dissociation of gene 32 protein from single-stranded DNA is very efficient and has interesting implications: it could provide a way to facilitate a rapid ”zippering” of the two complementary DNA strands during DNA replication and genetic recombination.