Viral vectors derived from adeno-associated virus (AAV) are rapidly becoming the vehicles of choice for gene therapy applications. AAV-2 is the adeno-associated virus serotype most commonly employed in AAV-mediated gene therapy studies; however, recently developed vectors derived from alternative serotypes of AAV, such as AAV-5, are receiving special attention due to their disparate tissue tropisms and potential for serial administration. In this report, we describe a rapid and efficient method for the serum-free production and column purification of recombinant AAV-5 particles. This method utilizes a combination of anion-exchange chromatography and gel filtration chromatography to purify recombinant AAV particles to near homogeneity. Importantly, viral particles are captured directly from cellular extracts with high efficiency, and vector purification is achieved in less than one working day with a minimal amount of sample manipulation. The method described in this report does not require partial purification by density centrifugation, detergent treatment, or solvent extraction to achieve efficient levels of column binding and vector purification.