Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity. Tissue distribution studies in mice using [(14)C]-labeled 3 indicate that adipose beds serve as ligand depots, contributing to the long terminal half-lives of these compounds.