We compared localization and distribution of putative cholinergic fibers by acetylcholinestersae and of adrenergic fibers visualized by the glyoxylic acid technique in the aganglionic segment using whole mount preparations of aganglionosis rat (AGR) and compared them with those of normal littermates. We also attempted simultaneous staining of acetylcholinesterase (AChE) and catecholamine fluorescence (C-F) on the same whole mount preparations to compare the differences in distribution pattern. All AGR used in this study had narrowed segments of the bowel extending from the distal ileum to the anus, and had no ganglion cells in these narrowed segments. In the intermuscular space, normally occupied with myenteric ganglion, of the narrowed distal colon and rectum, various sizes of nerve bundles and fibers reactive for AChE and C-F appeared to make coarse and irregular networks. These thick nerve bundles appeared to ascend to the proximal colon and disappeared in the cecum. In the distal ileum, almost totally absence of AChE positive nerve fibers, but a few fine C-F fibers, probably associated with blood vessels, were observed. By the method of simultaneous staining of AChE and C-F method in the whole mount prearations, the thick nerve bundles in the narrowed segments showed both of AChE positive and C-F positive. However, there were differences in peripheral fine nerve fibers in the segment ; especially numerous perivascular C-F positive nerve fibers, but a few AChE positive ones were found. In the upper aganglionic narrowed segments, greatly diminished numbers of AChE positive and C-F positive nerve fibers were found in the circular muscle layer and in the submucosal layer. In the lower aganglionic narrowed segments, there were thick nerve budles, forming irregular interlaced network. The role of these extrinsic nerve fibers in aganglionic segments is unclear.