Organoid cultured explants from differentiated tissues have gained renewed interest in the undertaking of physiological and pharmacological studies. In the present work, we examined the pharmaco-mechanical properties of an in vitro model, consisting of organoid cultured rings derived from rat extra- and intrapulmonary arteries, over a period of 4 days in culture. Male Wistar rats were humanely killed. The heart-lung block was removed, extra- and intrapulmonary arteries of the first and second order were dissected. Mechanical changes were quantified using isometric tension measurements on both fresh and cultured pulmonary arterial tissues, in the presence or absence of 10% fetal calf serum. Serum-free culture medium was supplemented with 1% insulin–transferrine–selenium (ITS). Conventional histochemical and immunofluorescent stainings were also performed to assess tissue structure integrity while the TUNEL method was used to detect apoptosis. Results are expressed as the mean ± S.E.M. with n indicating the number of rings used. Statistical analyses were performed using unpaired Student’s t tests as well as ANOVA for global comparisons of the curves. Values of P < 0.05 were considered significant. The explants developed spontaneous rhythmic contractions (SRC) in approximately half of the vessels. SRC amplitude and time course were significantly modified by conditions and agents acting on membrane potential, namely high potassium solutions or levcromakalim (0.1–1 μM), a potassium channel opener (n = 5–10). Nitrendipine, an L-type calcium channel blocker, suppressed SRC (n = 4–9). Cultured explants also developed a hyper-reactivity to high potassium challenges of 10–40 mM (n = 9–19). Whereas contraction to serotonin (5-HT) was enhanced in intrapulmonary arteries (n = 7–16), contraction to endothelin-1 remained unchanged after 4 days of culture (n = 10–18). Serum did not alter contractile properties during the culture period. Endothelial-dependent relaxation was maintained in response to A23187 (500 μM; n = 7–10) but was abolished in response to carbamylcholine (10 μM; n = 16–22). Histological analyses revealed the absence of hypertrophied vascular wall while the TUNEL technique attested to the absence of apoptosis. In conclusion, contractile phenotype and tissue structure integrity are well preserved during short-term culture of organoid explants making this model quite suitable for pathophysiological studies, such as the use of small interference RNA to study the functional role of specific proteins. Moreover, this organ culture model could be relevant in addressing the effect of chronic hypoxia in vitro.