We have examined the covalent chromatography of Lactobacillus casei thymidylate synthase on thiopropyl-Sepharose 6B resin. This enzyme is a dimer of identical subunits, each of which contains one active site that features a catalytic sulfhydryl group (Cys-198). Reversible coupling was achieved via the attack of one of the enzyme's two catalytic cysteine residues, causing displacement of 2-thiopyridone, the reactive moiety of the resin. To establish the usefulness of this matrix for immobilization and the conditions required for chromatography, model studies were conducted with 2,2'-dithiodipyridine. The chemical modification of thymidylate synthase with 2,2'-dithiodipyridine was shown to be specific for the catalytic sulfhydryl groups of the native dimer, titrating 1.51 sulfhydryl groups, while 2.93 cysteines were modified in the GnHCl-denatured protein. The former reaction, which resulted in total loss of enzyme activity, was reversible with complete recovery of control activity within 30 min after addition of 100 mM 2-mercaptoethanol. Characterization of the protein pools generated in the covalent chromatography procedure indicated that the enzyme fraction washing through the column without attachment had substantially lower catalytic and ligand binding activities than the original protein stock; conversely, the enzyme fraction eluted from the column by 2-mercaptoethanol exhibited higher levels of these activities. Gel electrophoresis studies further illustrated that the unique application of the covalent chromatography technique described herein fractionated homogeneous thymidylate synthase protein into enzyme pools exhibiting distinct biochemical properties. As immobilization reaction times were increased beyond 6 h, the coupling of thymidylate synthase was demonstrated to occur through more than one enzymic sulfhydryl group. Interestingly, no covalent coupling was detected in attempts using activated thiol-Sepharose 4B, a result underlining the importance of the structure of the resin linker arm in enzyme immobilization.