1. Reactive Blue 2 (Cibacron Blue 3G-A) is a competitive inhibitor of bovine heart cyclic nucleotide phosphodiesterase (K(i) 0.3mum). The K(i) increases with increasing temperature, suggesting that hydrophobic interactions are not largely responsible for the binding of the dye. Another 25 sulphonated aromatic dyes are also competitive inhibitors of the cyclic nucleotide phosphodiesterase (K(i) values in the range of 0.06-13.6mum). 2. These dyes (covalently linked to Dextran 40) inhibit bovine heart cyclic nucleotide phosphodiesterase. Reactive Blue 2 (covalently linked to Dextran 40) is a competitive inhibitor of the phosphodiesterase (K(i) 0.4mum). 3. Bovine heart cyclic nucleotide phosphodiesterase is retained on a column of Reactive Blue 2-Sephacryl S-200 and can be eluted from the column by 3':5'-cyclic AMP. 4. A variety of the dyes (either free or covalently linked to Dextran 40) are competitive inhibitors of rabbit muscle lactate dehydrogenase. 5. The effectiveness of a wide range of structurally dissimilar dyes as competitive inhibitors of lactate dehydrogenase and cyclic nucleotide phosphodiesterase compromises proposals for the use of Reactive Blue 2 as a specific probe for the dinucleotide-binding structural domain present in many dehydrogenases and kinases. Detailed information of the various dyes used has been deposited as Supplementary Publication SUP 50089 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.