The peptidyltransferase region of the 23S rRNA gene (the probable target site for the macrolides) was investigated in blood isolates of Mycobacterium avium recovered from 38 patients before and after the development of clarithromycin resistance. Point mutations were identified in 100% of the 74 resistant relapse blood isolates but in none of 69 susceptible pretreatment isolates. Multiple mutations were identified in isolates from 23 (61%) of 38 patients. Of the 63 identified mutations, 95% involved adenine at bp 2058. Single-colony clones from cultures that were mixtures of more than one mutation revealed a single mutation within each clone. Pulsed field gel electrophoresis of genomic DNA restriction fragments revealed that 13 (81%) of 16 multiple mutations identified in the same patient were derived from a single infecting strain. In vitro investigation revealed the same point mutations observed in vivo. This study defines the probable mechanism of clarithromycin resistance in M. avium and provides in vivo evidence that mutational resistance is random and selection-directed.