The addition of 5.6 mM crotonic acid to Streptomyces hygroscopicus var. ascomyceticus fermentations producing immunomycin reduced the level (as a percentage of immunomycin) of the major analog impurity L-683,795 from 6.7% for the control to 2.5%. Crotonic acid supplementation did not increase the titer of immunomycin and appeared to suppress production at higher concentrations. The addition of butyrate or valine, which can be catabolized to butyrate, at the concentrations used for crotonic acid were not as effective in reducing the percentage of L-683,795. Importantly, 5.6 mM crotonic acid supplementation reduced the L-683,795 level from 7.0% to 4.5% at the 23-l fermenter scale indicating that the effect of crotonic acid supplementation is scaleable to at least the laboratory bioreactor scale. Moreover, crotonyl-coenzyme A reductase (crotonyl-CoA reductase) activity could be detected in cell extracts. This work suggests that crotonic acid can be converted to butyrate through the activity of crotonyl-CoA reductase and can serve as a source of exogenously added C-4 precursor for macrolide biosynthesis.