D13 protein trimers, which form an external lattice providing curvature to the membrane of vaccinia virus immature virions, are the target of the drug rifampicin. We obtained 63 rifampicin-resistant mutants following random PCR mutagenesis of the D13L gene and 5 that arose spontaneously. Sequencing indicated that 26 mutants contained a single, unique, amino acid substitution whereas others contained 2 or more. The single mutations, including 6 previously identified, mapped to 24 different amino acids that were distributed over the length of the protein with the majority clustered between amino acids 17 to 33, 222 to 243 and 480 to 488. Two or three different substitutions occurred in six of the 24 amino acids. Representative mutant viruses of each cluster replicated to wild-type levels in the absence of rifampicin and nearly two logs higher than wild-type in the presence of drug. The large number and fitness of the mutations are remarkable in view of the extreme sequence conservation (99-100% amino acid identity amongst all orthopoxviruses). Clustering of mutations could suggest the presence of a rifampicin-binding pocket comprised of discontinuous regions of D13.