The responsiveness to transforming growth factor-β1 (TGF-β1) of two human keratinocyte cell lineages (FK16A and FK18B) generated after transfection with HPV-16 and HPV-18, respectively, was investigated. Both cell lineages revealed loss of heterozygosity (LOH) at 18q and/or 3p associated with the acquisition of the immortal phenotype. These loci harbour genes (TGF-β receptor II gene at 3p, and Smad2 and Smad4 genes at 18q) encoding products involved in the TGF-β1 signalling pathway. Mortal and early immortal stages of both cell lineages displayed growth reduction upon exposure to TGF-β1 concentrations in the range 100 pg/ml to 1 ng/ml. However, the late immortal stages were resistant to TGF-β1 at concentrations up to 10 ng/ml. TGF-β1 receptors type I and II were expressed at all stages in both cell lineages. Moreover, mRNA levels of Smad2 and Smad4 genes were nearly constant throughout. TGF-β1 expression and secretion, which were demonstrated in all analysed stages, may provide selective conditions underlying unresponsiveness to TGF-β1 upon prolonged monolayer culturing. Thus, LOH at 3p and/or 18q seen during HPV-mediated immortalization of human keratinocytes was not associated with resistance to TGF-β1-mediated growth inhibition or a marked reduction in TGF- β1 receptors and mRNA levels of Smad2 or Smad4. Therefore, alternative events are likely to underlie unresponsiveness to TGF- β1 in late-passage FK16A and FK18B cells.