The mechanism of anthelmintic resistance against the widely used macrocyclic lactones (MLs) is still not fully understood. Pharyngeal, somatic body muscles and the ovijector have been proposed as putative sites of action as well as resistance. In the present study the effects of three avermectins and three milbemycins on adult parasitic nematodes were evaluated in vitro. The Muscle Transducer system was used to investigate the effects of MLs on muscle contraction in female Haemonchus contortus and effects on motility were measured in Ostertagia (Teladorsagia) circumcincta using the Micromotility Meter. Concentration-response curves for all substances in both systems shifted to the right in the resistant isolates. Resistance was present to ivermectin (IVM) and its components IVM B1a and IVM B1b, suggesting that both components are involved in the mode of action and resistance. No consistent patterns of potency and resistance of the substances were observed except that milbemycins generally showed lower resistance ratios (RRs) than IVM. IVM and IVM B1b were the most potent inhibitors of contraction and motility in both susceptible isolates and also showed the highest RR in both species. Low RRs for milbemycins recorded in vitro for highly resistant isolates in vivo suggest that other factors such as pharmacokinetics influence drug potency in vivo.