Oligonucleotide probes targeting 16S ribosomal RNA were designed for species-specific identification of the porcine mycoplasmas Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma hyosynoviae using a fluorescent in situ hybridisation assay. The specificity of the probes was evaluated using pure cultures as well as porcine tissue sections with artificial presence of mycoplasma, and the probes were found specific for the target organisms. The assay was applied on sections of 28 tissue samples from pigs infected with one or more of the three Mycoplasma species as determined by cultivation. M. hyopneumoniae and M. hyorhinis were identified in accordance with cultivation in lung sections, from nine pigs affected by catarrhal to purulent bronchopneumonia. Likewise, in eight cases of fibrinous pericarditis, M. hyopneumoniae, M. hyorhinis and M. hyosynoviae were the infectious agents, according to cultivation and were correctly identified by in situ hybridisation. Out of I I joints cultivation positive for M. hyosynoviae, the probe was only able to identify M. hyosynoviae in eight cases probably due to a low number of microorganisms in the tissue sections. The in situ hybridisation assay is well suited for use in diagnostic and experimental work as well as a toot for pathogenesis studies.