In an accompanying paper (Kennedy, M. C., Spoto, G., Emptage, M. H., and Beinert, H. (1988) J. Biol. Chem. 263, 8190-8193), it was shown that one cysteine per mol of aconitase is modified by a variety of sulfhydryl reagents. We have identified the tryptic peptide that contains the iodoacetamide-reactive cysteine. We have also demonstrated that this cysteine is the primary site of modification by phenacyl bromide (2-bromoacetophenone), a spin label analogue of N-ethylmaleimide (HO-461) and iodoacetate in both the 3Fe and 4Fe forms of aconitase. The amino acid sequence of the peptide containing the reactive cysteine from beef heart aconitase shares no homology with the reactive cysteine-containing peptide reported for pig heart aconitase (Hahm, K.-S., Gawron, O., and Piszkiewicz, D. (1981) Biochim. Biophys. Acta 667, 457-461). We also report the amino acid compositions and sequences of seven other cysteine-containing tryptic peptides from beef heart aconitase. However, none of the cysteinyl peptides isolated were found to correspond to the reported pig heart reactive cysteinyl peptide. Evidence is also presented that no previously unreactive cysteine becomes exposed and reactive to sulfhydryl reagents in the conversion from the [4Fe-4S] cluster of the enzyme to the [3Fe-4S] cluster. We conclude from this that any potential cysteine ligand to the Fea site of the cluster must be inaccessible to solvent in the 3Fe form or, alternatively, that active 4Fe aconitase does not contain a cysteine ligand to the Fea site.