The specificity of the ATP-binding site of rhodopsin kinase was studied with adenosine analogues that are competitive inhibitors. Systematic changes in the ribose ring (position 5') and the purine ring (positions 2, 6, 7, 8, and 9) and determination of the inhibitory properties of these analogues lead to the following conclusions: (1) The N6 nitrogen in the purine ring is essential for binding at the active site, which may explain the marked preference for ATP rather than GTP as substrate. (2) The configuration of the sugar moiety is critical for the binding. (3) Positions 2, 3, and 8 of the purine ring, as well as the polyphosphate chain, play a minor role in substrate recognition by rhodopsin kinase. (4) ATP gamma S is a good substrate for rhodopsin kinase (thus rhodopsin phosphorothioate, a phosphatase-resistant product, can be formed in order to study the role of phosphorylation in rod outer segments). Pyrrolopyrimidine derivatives are very potent inhibitors of rhodopsin kinase. The Ki of one of these, sangivamycin, is 180 nM. Sangivamycin in solution assumes the anti conformation, as determined by nuclear Overhauser measurement. These measurements show that the most potent inhibitors of rhodopsin kinase, sangivamycin and toyocamycin, occur in solution preferentially in the anti conformation. Many nucleotides and nucleosides tested that are not inhibitors are syn, and many that are inhibitors form a mixture of syn and anti. The hypothesis that inhibitors may have a conformation intermediate between syn and anti was strengthened by testing a cyclic nucleoside locked in an anti conformation.(ABSTRACT TRUNCATED AT 250 WORDS)