The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.