Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1,4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1,4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.