Monoclonal antibodies (mAbs) raised against adult leech nervous systems were screened on embryos of the leech Haemopis marmorata in order to determine when in development specific antigens are first expressed and the order in which they are expressed by different cells or tissues. Three of the mAbs produced by Zipser and McKay (Zipser, B., and R. McKay (1981) Nature 289: 549-554) were screened: Lan3-1, Lan3-5, and Lan3-6. Each mAb shows a different pattern of labeling in the adult leech nerve cord (Zipser, B. (1982) J. Neurosci. 2: 1453-1464). The embryonic stages studied were from 5 days after egg deposition to 30 days (emergence from the cocoon). The pattern of labeling was assayed in whole mounts using horseradish peroxidase-conjugated second antibodies. The principal results are as follows. (1) Antigens recognized by Lan3-5 are first expressed by the glia of the roots of the anterior segmental ganglia at 6 to 7 days, several days later by the interganglionic connective glia, and near the end of embryonic development by ganglionic neurons. An anterior to posterior temporal gradient is observed in the expression of these antigens. In addition, Lan3-5 also labels the protonephridia and nephridia from early development onward. (2) Antigens recognized by Lan3-6 are first expressed by a pair of neurons in each segmental ganglion and later in development by additional neurons. By the time of emergence, however, only about half of the neurons that label in the adult have done so, implying that some neurons express these antigens postembryonically. Labeling with Lan3-6 is first seen in neuronal somata and only later in neuronal processes. (3) Antigens recognized by Lan3-1 and expressed by segmentally specific neurons in ganglia 5 and 6 are not detectable during embryonic development, but are so at early postembryonic stages. Thus, these three mAbs provide an approach to study different aspects of the development of the leech nervous system, specifically the relation between glial and neuronal differentiation and the genesis of segmentally specific phenotypes.