Rhodopsin kinase catalyzes the incorporation of up to seven phosphates into the carboxyl terminal region of freshly bleached rhodopsin. In order to study the mechanism of this reaction, we have separated different phosphorylated species of rhodopsin using Mono P FPLC chromatofocusing chromatography. The purity of the isolated species of rhodopsin was determined by isoelectric focusing. Separation yielded two forms of monophosphorylated and two diphosphorylated species of rhodopsin. Other species, containing up to five phosphates, were not fully separated. The phosphorylated forms of rhodopsin were characterized by competition enzyme-linked immunosorbent assay and immunoblotting using anti-rhodopsin site-specific monoclonal antibodies. A combination of the above methods allowed quantitative determination of the formation of different phosphorylated species of rhodopsin. A computer model for the consecutive time course of rhodopsin phosphorylation was developed and employed to characterize this reaction. Our data suggest that the rate of incorporation of the first phosphates into rhodopsin is slower than the rate of formation of more highly phosphorylated species. These data are supported by results showing that some monophosphorylated synthetic peptides are phosphorylated significantly faster than control unphosphorylated peptides.