Previously, we reported successful immortalisation following hTERT introduction in primary human fibroblasts, strain VH25. Since one subclone in that study developed some abnormalities, we decided to study eight additional independent immortalised clones to get an indication of the frequency and type of abnormalities that develop after hTERT-mediated immortalisation. We show that although some cell lines can maintain a normal phenotype for 500 population doublings (PDs), in four clones after 150–300 PDs changes developed in basal and radiation-induced p53 and p21WAF-1,CIP-1 levels. Our experiments demonstrate that, after prolonged culture, cells with abnormalities in cell cycle control parameters can take over the population. This calls for caution when working with hTERT-immortalised cells in vitro as well as in vivo.