We have recently cloned cDNAs encoding two invertase inhibitors from Nicotiana tabacum, Nt-inh1, an apoplastic isoform, and Nt-inhh, a vacuolar isoform (Greiner et al. (1998) Plant Phys. 116, 733–742; Greiner et al. (1999) Nature/Biotech. 17, 708–711). A database search revealed the presence of related sequences in other dicot families. Here we report the presence of Nt-inh1-related proteins (INH-RPs) in apoplastic fractions from Chenopodium rubrum and Daucus carota suspension-culture cells. Furthermore, we demonstrate that, in Lycopersicon esculentum, the expression of INH-RPs is highly regulated during plant development. In immature tomato fruits two INH-RP isoforms are expressed, whereas in mature fruit a single isoform is detected. Sequential extraction of apoplastic and intracellular fractions from mature fruit pericarp tissue revealed that the major portion of invertase and INH-RP are localized in the vacuole. Recovery of the non-glycosylated INH-RP with the glycosylated invertase from the Concanavalin A-bound fraction indicates that INH-RP forms a stable complex with vacuolar invertase. As Nt-inh1 and INH-RP from different species contain four conserved cysteine residues, we have compared the inhibitory activity of oxidized and dithiothreitol (DTT)-treated recombinant Nt-inh1 protein. Only the oxidized form is active as an invertase inhibitor. Its higher mobility during sodium dodecyl sulfate–gel electrophoresis, as compared to DTT-treated Nt-inh1, suggests that disulfide bridge(s) prevent the inhibitor from unfolding. A mechanism is proposed for the post-translational inactivation of cell wall and vacuolar invertases via invertase inhibitors during critical stages of plant development.