Proteomics studies have identified SPAK and OSR1 in exosomes isolated from body fluids such as blood, saliva, urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immuno-gold analyses that these exosomes express SPAK, OSR1, and NKCC1. We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell to cell communication vessels. Similarly, co-culturing cells expressing different fluorescently-tagged proteins resulted into the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.