The alpha-helices of coiled-coil proteins are predominantly parallel, in contrast to the general preference for an antiparallel orientation of interacting alpha-helices found in globular proteins. One intriguing exception is the antiparallel, two-stranded coiled coil comprising the long helical arm of the bacterial seryl tRNA synthetases (SRS). A recombinant 82-residue peptide corresponding to the helical arm of Escherichia coli SRS folds into a stable, monomeric, helical structure in the absence of the rest of the protein, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. However, peptides corresponding to the individual helices of SRS are unstructured at neutral pH and do not associate appreciably at total peptide concentrations up to 100 microM. Covalent attachment of the the two peptides through a nonnatural, disulfide-containing linker restores structure and allows study of variants in which the individual helices are constrained to interact in either an antiparallel or a parallel orientation. We find that the antiparallel species are substantially more helical and more stable to thermal denaturation than their parallel counterpart. Thus, the SRS helical arm is an autonomously folding unit, and, unlike most other coiled coils, has an intrinsic preference for an antiparallel orientation of its constituent helices.