rCAS is a recombinant Constant And Subrepeat (rCAS) protein modelled after tandem core repeats found in a 1000-kDa silk protein synthesized by larvae of the midge, Chironomus tentans. rCAS is encoded by a synthetic gene (synCAS) which is expressed in bacteria. Purified rCAS has four cysteine residues that participate in formation of two intramolecular disulfide bonds. Here we report the results of amino acid sequencing and electrospray ionization mass spectroscopic analyses of tryptic fragments of native and reduced rCAS which suggest that these disulfide bonds are heterogeneous. To assist in studying the formation of disulfide bonds in reduced and refolded rCAS, a series of synCAS mutants were constructed with cysteine to alanine substitutions. In comparison to wild-type rCAS and a Cys79→Ala mutant, the refolding of Cys9→Ala appears to be partially inhibited.