Abstract Human myocardial fibroblasts (HMF) have proved to be useful as a species specific cell culture system in various studies on myocarditis and cardiac remodelling. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary HMF with the E6 and E7 genes of the oncogenic human papillomavirus (HPV) 16. Successful transfection was demonstrated in 3 of 12 experiments by detection of E6–E7 gene transcription with nucleic acid sequence based amplification (NASBA). No significant change of phenotype was noted in the emerging cell lines (HMF 1226D, HMF 1321D, HMF 1226K), but their in vitro lifespan was increased by 20 to 30 population doublings until cells entered crisis. A single subclone of HMF 1226K had a transformed phenotype and continued to proliferate indefinitely. This subclone (HMF 1226K/I) was considered to be immortalized and telomerase activity was detected. Despite the increased risk of mutations due to abrogation of p53 function, HMF 1226K/I and the HMF lines with an increased lifespan retained the properties of primary HMF cells, as they expressed fibroblast markers (prolyl-4-hydroxylase, vimentin), cytokines (interleukin 1α, 6, 8), and angiotensin II receptors and still were permissive for coxsackievirus B3 infection.