North American pawpaw [Asimina triloba (L.) Dunal] is a temperate climate member of the Annonaceae used as an ornamental and specialty tree fruit crop. Micropropagation could provide a clonal propagation method for plant production and contribute to germplasm preservation. A pawpaw culture was established that has produced shoot-bud clusters for over six years when maintained on a plant growth regulator (PGR) medium with BA (8.9 µM) + NAA (2.3 µM). Shoots on PGR medium failed to elongate beyond 2 cm compared to approximately 12% for shoots on PGR-free medium. Single shoot-buds subcultured three times to PGR-free medium continued to initiate between 5 to 7 shoot-buds per culture and shoot elongation remained at approximately 16%. This suggests that these pawpaw cultures had become habituated to autonomously produce shoots without cytokinin and auxin induction. The treatment that promoted the greatest shoot elongation (49%) was the combination of PGR-free medium plus activated charcoal in either the agar medium alone or a 3-day liquid overlay containing activated charcoal. Numerous attempts to root elongated microcuttings were unsuccessful. The same mechanism that makes pawpaw cultures habituated for autonomous shoot production probably contributes to the inability of microcuttings to root.