The ability to accumulate osmoprotectant compounds, such as betaines, is an important evolutionary feature in many organisms. This is particularly the case for organisms that live in variable environments, which may have fluctuations in moisture and salinity levels. There is, surprisingly, very little known about betaines in soil invertebrates in general, and there is almost no information about earthworms a group that are important 'ecosystem engineers' and key indicators of soil health. Here, we describe a fast and reliable H-1-C-13 heteronuclear single quantum coherence (HSQC) 2D NMR approach for the metabolic profiling of a series of betaines and related metabolites in tissue extracts, and list H-1 and C-13 chemical shifts for the trimethylammonium signal for 23 such compounds. The analysis of ten different species from three different families (Lumbricidae, Megascolecidae and Glossoscolecidae) showed an unexpected diversity of betaines present in earthworms. In total ten betaines were identified, including hydroxyproline-betaine, proline-betaine, taurine-betaine, GABA-betaine and histidine-betaine, and a further eleven as-yet unassigned putative betaine metabolites detected. The findings clearly indicate a hitherto-unappreciated important role for betaine metabolism in earthworms. (C) 2012 Elsevier Inc. All rights reserved.