In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.