ABSTRACT-Spontaneous tone of longitudinal muscle in guinea pig ileum was investigated in three types of preparations, intact segment preparation, segment preparation deprived of the mucosal layer and longitudinal strip preparation. Tone was defined as the sustained contraction that was lost by 10-7 M atropine in the isotonically recorded manner. The magnitudes of tone were constant for at least 3 hr in the two types of segment preparations. Contractions in response to 10-6 M acetylcholine, which were induced 9 times with an interval of 20 min between each induction, were almost identical throughout the period. In the longitudinal strip preparation, on the other hand, the tone gradually decayed and was eventually lost, while the amplitudes of acetylcholine-induced contractions were reciprocally increased. The tone in the intact segment preparation was reduced to 19% of the control by tetrodotoxin (3 x 10-7 M), to 51 % by indomethacin (3 x 10-6 M) and to 26% by 1V6-cyclopentyladenosine (10-7 M), but was not affected by AA-861 (3 x 10-6 M) or CP-96,345 (3 x 10-7 M). In the three types of preparations, the dose-response curves for acetylcholine were alike with similar EC50s. These results suggest that the tone of longitudinal muscle was mainly induced due to neural activity in the myenteric plexus of guinea pig ileum and that sensitivity to acetylcholine was not affected by the neural activity.