A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (vol/vol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain.