Interferons (IFNs) are naturally produced cytokines with multiple important biological functions. The activity of recombinant feline IFN-omega (rFeIFN-omega) alone and in combination with chemotherapeutic drugs was tested on canine and feline mammary carcinoma cell lines (REM134 and CAT-MT) and putative tumour-initiating cells (TIC) derived from these cell lines by sphere assay. Viability was measured by chemoluminescence and a one-way analysis of variance and Student's t-tests were used for statistical analysis. rFeIFN-omega showed in vitro antitumour activity on feline and canine mammary carcinoma cells and putative TICs with a dose-dependent and target cell-specific action. Putative TICs were more resistant to the action of rFeIFN-omega compared with daughter REM134 and CAT-MT cells. REM134 cells and TICs were more sensitive to rFeIFN-omega compared with the feline counterparts. An additive effect was noticed between rFeIFN-omega and conventional anticancer drugs, in particular following co-culture of cells with anthracycline drugs. The results suggest that rFeIFN-omega warrants further investigation as a therapeutic adjunct in feline and canine mammary tumours.