A novel and highly specific method has been developed for the determination of dimethyl sulfoxide (DMSO) at nanomolar levels in aqueous solution. After removal of dimethyl sulfide (DMS) from the solution by purging with oxygen-free nitrogen, DMSO is reduced to DMS using the enzyme DMSO reductase purified from the bacterium Rhodobacter capsulatus. The resulting DMS is cryogenically trapped and analyzed by gas chromatography. The detection limit is 0.016 nmol of DMSO per sample (maximum volume 100 cm3), and precision for standards in the concentration range 0.063–1.0 nmol of DMSO is within 2%. The high specificity of the enzymatic reduction step for DMSO was proven in tests with a range of organic sulfur compounds including dimethyl sulfoniopropionate (DMSP, the major biochemical precursor of DMS in algae). The possibility of competitive inhibition of the enzyme was also addressed. In comparison to existing methods for DMSO analysis, the enzymatic method is advantageous in terms of its specificity, its ease of use, and the safe nature of the reducing agent. The technique can be used for freshwater and seawater samples and may also be suitable for pharmaceutical, food, and beverage industry applications. Depth profiles of DMSO, DMS, and DMSP for seawater samples collected in the Pacific Ocean are presented.