Tritiated thymidine (3H-TdR) injected before a stab wound of the spinal cord or transection of the hypoglossal nerve has resulted in many labeled reactive cells in the CNS after injury, most of which have the ultrastructural features of microglia. To test for the possible origin of these labeled cells from monocytes, we examined them for the presence of sodium fluoride- (NaF) sensitive non-specific esterase (NSE), an enzyme characteristic of monocytes. Some of the labeled cells in stab wounds had NaF-sensitive NSE, but no such cells were found in the nucleus of the injured hypoglossal nerve. To test for the possibility that the NSE-negative labeled cells had been labeled by reutilization of 3H-TdR, we used 125I-5-iodo-2'-deoxyuridine (125I-UdR), a thymidine analogue with a much lower rate of reutilization, to label blood mononuclear cells prior to either a spinal cord stab wound or hypoglossal axotomy. The number of labeled cells was decreased in the spinal cord wound, but more than half were NSE-negative. No labeled blood mononuclear cells were found in the hypoglossal nucleus, although there was no decrease in the hyperplasia of unlabeled non-neuronal cells. When 125I-UdR was injected on the fourth day after hypoglossal axotomy, or when both 3H-TdR and 125I-UdR were injected simultaneously before hypoglossal axotomy, many labeled cells were found in the hypoglossal nucleus, indicating that 125I-UdR can be used by the reactive cells and that it did not inhibit their proliferation. Therefore, the microglial cells that proliferate in response to peripheral nerve injury are not recently derived from any type of circulating large blood mononuclear cell. The most likely explanation for the presence of the 3H-TdR-labeled cells in the nucleus of the injured hypoglossal nerve is that they were proliferating intrinsic cells labeled by reutilization of 3H-TdR.