The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-[1-(6-aminohexyl)-levulinic acid amide]-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 126.96.36.199) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.