A fluorescence immune assay designed to measure anti-nucleotide antibody activity is described based on the synthesis of a fluorescent nucleotide probe possessing a low fluorescence quantum yield when free in aqueous solution (neutral pH). The fluorophore, AmNS (1-naphthylamine-5-sulfonic acid), was covalently conjugated to various phosphate derivatives of nucleotides through carbodiimide activation to form the fluorescent probe. The quantum yield (phi) of the fluorescent nucleotide in solution (neutral pH) was approximately 0.025 based on an excitation maximum of 320 nm and an emission maximum of 460 nm. Anti-nucleotide antibodies elicited in rabbits and mice served as standard immunological reagents in development of the fluorescence assay. Upon binding of an AmNS-nucleotide conjugate with homologous anti-nucleotide antibodies, the fluorescence quantum yield of the conjugate was significantly enhanced (12-35 x). Fluorescence enhancement was not obtained upon incubation of the fluorescent probe with normal Ig, non-immune rabbit sera and murine ascites fluid, or bovine and rabbit serum albumin. Nucleotide inhibition reactions were quantitatively measured in the fluorescence assay. Nucleotide binding results obtained with the fluorescence assay were correlated with a modified radioimmune Farr assay.