<jats:title>Abstract</jats:title><jats:p>Avermectin and its analogues are produced by the actinomycete <jats:italic>Streptomyces avermitilis</jats:italic> and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC‐B1), which is sold commercially as Dectomax™, is co‐produced during fermentation with the undesired analogue CHC‐B2 at a CHC‐B2:CHC‐B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The <jats:italic>aveC</jats:italic> gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site‐specific mutagenesis and error‐prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC‐B1, produced relative to the undesired CHC‐B2 fermentation component. High‐throughput (HTP) screening of cultures grown on solid‐phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An <jats:italic>aveC</jats:italic> gene with mutations that result in a 4‐fold improvement in the ratio of doramectin to CHC‐B2 was identified. Subsequent integration of the enhanced <jats:italic>aveC</jats:italic> gene into the chromosome of the <jats:italic>S. avermitilis</jats:italic> production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 359–369, 2003.