Previous reports have demonstrated that aconitase has a single reactive sulfhydryl at or near the active site (Johnson, P. G., Waheed, A., Jones, L., Glaid, A. J., and Gawron, O. (1977) Biochem. Biophys. Res. Commun. 74, 384-389). On the basis of experiments with phenacyl bromide in which enzyme activity was abolished while substrate afforded protection, it was concluded that this group was an essential sulfhydryl. We have further examined the reactivity of this group and confirmed the result that, when reagents with bulky groups (e.g. N-ethylmaleimide or phenacyl bromide) modify the protein at the reactive sulfhydryl, activity is lost. However, when smaller groups, e.g. the SCH3 from methylmethanethiosulfonate or the CH2CONH2 from iodoacetamide, are introduced, there is only partial (50%) or no loss of activity. Experiments were performed to obtain evidence that these reagents are modifying the same residue. Methylmethanethio-sulfonate-treated enzyme showed an increase in the Km for citrate from 200 to 330 microM. EPR spectra were taken of the reduced N-ethylmaleimide- and iodoacetamide-modified enzyme in the presence of substrate. The former gave a spectrum typical of the substrate-free enzyme, while the spectrum of the latter was identical to enzyme with bound substrate. We, therefore, conclude that modification of this sulfhydryl affects activity by interfering with the binding of substrate to the active site and is not essential in the catalytic process.