Juvenile hormone (JH)-I and -III were used as model substrates to study the in vitro metabolism of JH in the hemolymph and body homogenates of the American dog tick, Dermacentor variabilis (Say). Ester hydrolysis was the principal pathway of JH metabolism in hemolymph and homogenates. JH also was converted into JH-diol primarily by body homogenates, indicating the presence of JH epoxide hydrolase activity. JH epoxide hydrolase activity, alpha-naphthyl acetate esterase activity, and protein concentration per milligram wet weight were significantly lower (t test, alpha = 0.05) in homogenates of partially fed, virgin and replete, mated females of D. variabilis compared with unfed, virgin females. The decline in these factors was probably because of the influx of water into the tissues caused by the blood meal. In addition, the epoxide hydrolase and alpha-naphthyl acetate esterase activity per milligram tissue protein decreased significantly during this time. Mating of fed females rather than feeding alone caused a significant decline in the tissue JH esterase activity per milligram wet weight but not per milligram protein. The JH esterase activity per milligram protein was significantly higher in partially fed, virgin and replete, mated females compared with unfed females, indicating that feeding may actually increase JH esterase activity on a protein basis. JH-III was metabolized 1.4 times faster than JH-I by the hemolymph of partially fed, virgin females. The inhibitors O,O-diisopropyl phosphorofluoridate and octylthio-1,1,1-trifluoropropan-2-one at 10(-4) M inhibited JH and alpha-naphthyl acetate esterase activity in hemolymph and body homogenate.