The North American pawpaw [ Asimina triloba (L.) Dunal] is a tree fruit in the early stages of commercial production in the United States. This plant contains annonaceous acetogenins in the twigs, unripe fruit, seeds, roots, and bark tissues, which display antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects, suggesting many potentially useful applications. However, commercial development of these compounds, based on twig extracts, has been problematic due to limited availability of biomass for extraction. Additionally, acetogenin compounds contained in fruit of pawpaw relatives (soursop or Annona muricata ) and tea made from the leaves of these plants may lead to an increased risk of atypical Parkinsonism later in life with overconsumption of these compounds. Therefore, the objectives of this study were (1) to determine if extracts of ripe pawpaw fruit pulp displayed acetogenin activity, (2) to identify potential acetogenin compounds in the fruit tissue, and (3) to determine if the acetogenin activity varied in diverse pawpaw genotypes and closely related Annona species. Extracts of ripe fruit had total extract weights and bioactivity using the brine shrimp bioassay similar to those from 'NC-1' pawpaw twig tissue. Pulp from soursop, cherimoya, and several additional pawpaw cultivars ('Mitchell', 'Overleese', 'NC-1','Zimmerman', 'Wells', and 'Sunflower') also displayed bioactivity, but peach or banana pulp did not. Ripe pawpaw pulp extract subjected to HPLC-MS analysis identified three prominent acetogenins: asimicin, bullatacin, and bullatalicin. This study points to pawpaw fruit pulp serving as a new biomass source for the extraction of acetogenin compounds for product development. An assessment of the potential human health risk of overconsumption of fruit and acetogenin bioavailability and degradation studies should be pursued.