Taxonomic studies were performed on a phenotypically homogeneous group of 13 mycobacteria isolated from clinical, veterinary and stream-water samples. The methods applied included chromatographic analyses of bacterial lipids, biochemical tests and sequencing of the 16S rDNA and the internal transcribed spacer 1 (ITS1) region. Positive results in urease, Tween 80 hydrolysis and pyrazinamidase tests and a negative result in a semi-quantitative catalase test, combined with the ability to grow at 42 degrees C, distinguished this group among the yellow-pigmented, slowly growing mycobacteria. Unique fatty acid and mycolic acid profiles in chromatographic analyses and the results of gene sequencing indicated that the novel isolates represent a previously undescribed species, for which the name Mycobacterium palustre sp. nov. is proposed. The fatty acid profile obtained by GLC was characterized by the presence of several methyl-branched fatty acid markers. The most prominent markers were 2-methyleicosanoic, tetracosanoic and hexacosanoic acids. According to 16S rDNA sequencing, M. palustre is phylogenetically closest to Mycobacterium kubicae, a recently described species. M. palustre gives a false-positive result in a hybridization test with the AccuProbe Mycobacterium avium complex. One of the strains was isolated from a lymph-node biopsy from a child with cervical lymphadenitis. Thus, M. palustre should be listed among potential inducers of paediatric lymphadenitis. The veterinary isolates originated from the lymph nodes of slaughter pigs. The majority of the strains were recovered from natural waters, which highlights the role of the environment as a source of potentially pathogenic mycobacteria. The type strain of M. palustre is strain E846T (= DSM 44572T = ATCC BAA-377T).