We used immunohistochemistry to investigate the colocalization pattern of calbindin D28k (Calb) with nitric oxide and acetylcholine in myenteric neurons in the rat esophagus and compared it to that in the ileum or distal colon. The proportion of Calb-immunoreactive (IR) neurons to the total neurons in the esophagus (8%) was lower than that in the ileum (38%) or distal colon (27%). A majority (84%) of the esophageal Calb-IR neurons were uniaxonal neurons. On the other hand, 88% and 66% of Calb-IR neurons in the ileum and distal colon, respectively, had Dogiel type II morphology, while most of the others were Dogiel type I neurons. Double immunolabeling indicated that most (87%) of the esophageal Calb-IR neurons were nitric oxide synthase (NOS) positive and a minority (21%) were choline acetyltransferase (ChAT) positive. Most (93% and 89%, respectively) of the Calb-IR neurons in the ileum and distal colon showed ChAT immunoreactivity and only a small number exhibited NOS immunoreactivity in the ileum and distal colon. In the esophagus, some of Calb-IR nerve endings surrounding the myenteric neurons were NOS positive, but no Calb immunoreactivity was found on the motor endplates of the striated muscles. Therefore, the present study revealed that most of the Calb-IR neurons in the esophagus are nitrergic, and it suggested that the Calb-IR neurons might be primarily involved in interneuronal roles in the esophageal nervous system.