To confirm the larvicidal activity of 2-alkyl fatty acids occurring in the overcrowding factors of mosquito larvae, and to investigate the relationship of the alkyl substitution on the carbon chain, 43 fatty acids were tested against first instars ofCulex pipiens quinquefasciatusSay.2-ethyloctadecanoic acid, 3-methyloctadecanoic acid, and 2,3-dimethyl-octadecanoic acid were the most active materials among those tested. Their activity as determined by LC50was in the range of 0.4–0.6 ppm.The larvicidal activity of the branched fatty acids was best correlated with the steric hindrance effect of 2– and 3-alkyl fatty acids on enzymatic esterification in vitro. The characteristic timing of larval death occurring immediately after ecdysis suggests that the antigrowth activity of these branched fatty acids results in the formation of new but water-permeable cuticle in mosquito larvae during ecdysis.