Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces coelicolor A3(2). One (strain 27) was analyzed in detail. Mutant 27 had a reduced ADP-ribosyl-transferase activity, exhibited substantial changes from the wild type in ADP-ribosylated protein profile during cell aging, and was defective in producing aerial mycelium and antibiotics. A 92-kDa ADP-ribosylated protein disappeared at the onset of differentiation in the parent strain but was present in mutant 27. Four ADP-ribosylated proteins (39, 41, 43, and 46 kDa) appeared at the onset of differentiation in the parent strain but were missing in mutant 27. Failure to ADP-ribosylate these four proteins was detected when the parent strain was grown in the presence of subinhibitory amounts of 3-aminobenzamide. Genetic analysis showed that the mutation, named brgA, conferring resistance to 3-aminobenzamide, cosegregated with the altered phenotypes (i.e., defects in ADP-ribosylation and aerial mycelium formation) and was mapped to a new locus near uraA. The brgA mutants were nonconditionally deficient in producing aerial mycelium and antibiotics, as determined by using various media, and had a morphological and physiological phenotype quite different from that of a bldG mutant carrying a mutation which was previously mapped near uraA. Among the known bld mutants, bldA, bldD, and bldG mutants exhibited a ADP-ribosylated protein profile similar to that of the wild type, while like mutant 27, bldB, bldC, and bldH mutants failed to ADP-ribosylate certain proteins.