The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane.