At two occasions (2004 and 2006), a similar cluster of culturable bacteria was found to be selected in the mycosphere of the basidiomycetous fungus Laccaria proxima ( Agaricales, Tricholomataceae) in the field. The bacteria, identified as related to Variovorax paradoxus, comprised 7.3–9% of the total culturable bacterial community in the L. proxima mycosphere, but were not found in corresponding bulk soil (<0.3%). One strain, denoted HB44, was selected in order to unravel the basis of the V. paradoxus mycosphere competence in in vitro experiments with the former Laccaria laccata, which was recently reclassified as Lyophyllum sp. strain Karsten ( Agaricales, Tricholomataceae). In liquid culture experiments, L. strain Karsten was shown to be an avid producer of glycerol, next to acetate and formate, which constituted the most abundant carbonaceous compounds released. Strain HB44 was able to grow avidly at the expense of the glycerol liberated by the fungus, as evidenced by proton NMR analysis of the fungal exudates in the medium before and after bacterial growth. In sterilized field soil, strain HB44 survived significantly better in the presence than in the absence of L. strain Karsten. Addition of a glycerol series to the sterilized soil (without the fungus) resulted in the persistence or growth of strain HB44, but only if the pH of this soil was previously set at 5.5. Thus, we provide evidence for the contention that tricholomataceous fungi can create specific niches in soil for the V. paradoxus-related strain HB44 and that glycerol may be a main carbon source that drives the selection of this organism.