The pesticide toxaphene with a total worldwide production of 1.3 Mt is found in the aquatic environment with a widespread, global distribution. Its analysis is difficult due to its complex character. A technical toxaphene mixture and a number of biological samples were analyzed by multidimensional gas chromatography (MDGC). Four different column combinations were used: DB5/DX4, DB5/FFAP, Ultra 2/DX4, and Ultra 2/Rtx 2330. The technical mixture consists of at least 246 congeners, while only 107 peaks were found in a one- dimensional GC/ECD chromatogram. The major peaks present in the technical mixture and biological samples mostly consist of 2-10 toxaphene congeners. These results show that in most cases single-column chromatography is not suitable for a congener-specific determination of the toxaphene congeners. The Rtx 2330 column is recommended as the best choice as a second column in MDGC of toxaphene, particularly because of its stability at higher temperatures and because of a slightly better separation, although the differences in results obtained with the different column combinations were relatively small. The relatively high concentrations of toxaphene in European fish, which may even lead to exceeding the tolerance levels for human consumption, and in marine mammals demand a proper monitoring of total toxaphene and individual CHBs in relevant samples from European aquatic ecosystems.