Abstract Intracellular distribution of the sialidase of adult bovine brain gray matter was determined. The sialidase was found to be concentrated in the synaptosome fraction, and when the latter was disrupted and further fractionated, sialidase activity was found to be concentrated in the synaptosomal membrane. Sialidase in all cell fractions showed activity toward an intrinsic endogenous substrate, as well as toward added disialo- and trisialogangliosides, and sialyllactose. Km values for the sialidase in an acetone powder preparation from gross gray matter were 8 x 10-5 and 2 x 10-3 m for gangliosides and sialyllactose, respectively. There was an unequivocal lag phase of about 15 min between the time of addition of exogenous substrate and the time the sialidase measurably acted on it. The enzyme was found to act on the intrinsic endogenous substrate during the lag phase. Thin layer chromatography showed monosialo-, disialo-, and trisialogangliosides to be present in all cell fractions having sialidase activity. Analysis of the active fractions after incubation at 37° for 75 min without added exogenous substrate showed that monosialoganglioside, the end product of enzyme action on the intrinsic ganglioside substrate, remained associated with the membrane fragments.