The intron-encoded U16 small nucleolar RNA (snoRNA) is a component of a new family of molecules which originate by processing of pre-mRNA in which they are contained. The mechanism of U16 snoRNA biosynthesis involves an initial step of endonucleolytic cleavage of the pre-mRNA with the release of a pre-snoRNA molecule; the subsequent step consists of exonucleolytic trimming that produces mature U16 molecules. In order to identify the molecular components involved in this peculiar biosynthetic pathway, we have undertaken the characterization of the endonucleolytic activity by biochemical fractionation of Xenopus laevis oocyte nuclear extract. In this paper we show the production of a protein fraction (BSF) which is highly enriched for a specific endonucleolytic activity that exactly reproduces the cleavage pattern of the U16-containing pre-mRNA identified in vivo in X. laevis oocytes and in unfractionated nuclear extract.