Bovine rhodopsin has been phosphorylated in rod outer segments by ATP and endogenous rhodopsin kinase. Mono-, di-, and triphosphorylated rhodopsins have been prepared by chromatofocusing. Nearly all of the phosphate is found in peptide 330-348, formed by digestion of phosphorhodopsins with endoproteinase Asp-N. Sequence analysis of the phosphopeptides shows that monophosphorylated rhodopsin consists of a mixture containing rhodopsins phosphorylated at 338Ser and 343Ser. Diphosphorylated rhodopsin is phosphorylated at both 338Ser and 343Ser. When rhodopsin becomes triphosphorylated it does not become phosphorylated on 334Ser but appears to become phosphorylated on one or more of the four threonine residues: 335Thr, 336Thr, 340Thr, and 342Thr.