The anthelmintic avermectin complex produced by Streptomyces avermitilis comprises major components (Ala, A2a, Bla, B2a) and minor b-components (Alb, A2b, Bib, B2b), with Bla and B2a being the most effective. This study aimed to develop a selective producer of only these two components. Mutagenesis of parent strain K139 using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) yielded mutant K2034 (aveD), which lacks avermectin B2 5-O-methyltransferase activity and accumulates B components (Bla, B2a, Bib, B2b). Another mutant, K2021 (X), was obtained that produces only a-components (Ala, A2a, Bla, B2a) due to drastically reduced incorporation of L-valine or its keto acid (2-oxoisovalerate). Protoplast fusion of K2021 and K2034 generated recombinant strains (e.g., K2038) with both phenotypes, which produce only Bla and B2a. Recombinants were efficiently isolated at a frequency of about 10^-2 and produced approximately 0.2 g/liter of avermectins. Genetic analysis suggested the loci of the two mutations (affecting methyltransferase activity and branched-chain keto acid incorporation) are sufficiently distant on the chromosome.