Aquaporin-4 (AQP4) is known to have two main isoforms M1 and M23 in the brain. Immunoblot analyses have provided evidence of additional AQP4 immunopositive bands, suggesting that the repertoire of AQP4 isoforms is broader than previously assumed. As isoforms beyond M1 and M23 are not observed in recombinant systems, investigation of novel isoforms requires the use of a native source. Here we report purification of AQP4 to three silver-stained proteins on SDS-PAGE. This was achieved by organelle separation, alkaline stripping of cellular membranes, detergent solubilization and multiple chromatographic steps. The three proteins that co-purified were identified as AQP4 by mass spectrometry. These results represent the first purification of AQP4 from a native source and demonstrate by mass spectrometry the presence of a third AQP4 isoform of 36 kDa in the rat brain. Immunoblots revealed that the same isoform is present in the mouse, pig, and human brain.