The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx--the RNAP "switch-region SW1/SW2 subregion"--is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here we report the frequency, spectrum, and fitness costs of Myx-resistance in Staphylococcus aureus. The resistance rate for Myx is 4-7×10(-8) per generation, which is equal within error to the resistance rate for Rif (3-10×10(-8) per generation). Substitutions conferring Myx-resistance were obtained in RNAP β subunit (six substitutions: V1080[1275]I, V1080[1275]L, E1084[1279]K, D1101[1296]E, S1127[1322]L, and S1127[1322]P) and RNAP β' subunit (five substitutions: K334[345]N, T925[917]K, T925[917]R, G1172[1354]C, and G1172[1354]D) (residues numbered as in S. aureus RNAP and, in brackets, as in Escherichia coli RNAP). Sites of substitutions conferring Myx-resistance map to the RNAP switch region SW1/SW2 subregion and do not overlap the binding site on RNAP for Rif, and, correspondingly, Myx-resistant mutants exhibit no cross-resistance to Rif. All substitutions conferring Myx-resistance exhibit significant fitness costs (4-15% per generation). In contrast, at least three substitutions conferring Rif-resistance exhibit no fitness costs (0% per generation). The observation that all Myx-resistant mutants have significant fitness costs whereas at least three Rif-resistant mutants have no fitness costs, together with the previously established inverse correlation between fitness cost and clinical prevalence, suggests that Myx-resistance is likely to have lower clinical prevalence than Rif-resistance.