Trichomonads are a group of anaerobic flagellates. Two species of intestinal trichomonads have been described in cats - pathogenic Tritrichomonas foetus and probably harmless Pentatrichomonas hominis. InPouch™ TF-Feline medium (Bio-Med Diagnostics, White City, Oregon, USA) is considered the gold standard for diagnosis of T. foetus infection in cats. It is commercially available, relatively cheap and easy-to-use. The medium is intended to be highly specific to T. foetus as morphologically similar Pentatrichomonas hominis and Giardia sp. do not survive here longer than 24h. In this study we successfully cultivated P. hominis in InPouch™ TF-Feline medium for 3 days after inoculation with cat faeces. The identity of the organism was assessed by sequencing of SSU rDNA and ITS region. Possible coinfection with T. foetus was ruled out using Tritrichomonas-specific PCR. Our results suggest possible misdiagnosis of tritrichomonosis in cats using InPouch™ TF-Feline medium. PCR-based verification of culture-positive samples prior the potentially neurotoxic ronidazole treatment is recommended.