The experimentally derived parameter Tm-t (tissue Tm) was defined previously to describe the end-point used for evaluation of the stringency of non-isotopic in situ hybridization and was found to differ from the theoretical melting temperature (Tm) for several HPV types. In this paper, the reasons for this discrepancy were investigated by performing a series of experiments with a variety of probes for both human genomic and integrated viral sequences in isolated and cultured normal and abnormal cells in addition to paraffin-embedded material. Tm-t was shown to be dependent on several parameters of probe and target, and on the sensitivity of the detection system used but was not affected by aldehyde fixation or paraffin wax embedding under optimal conditions of nucleic acid unmasking. These data support the hypothesis that differences between Tm-t and Tm may be due to the use of a different end-point for in situ hybridization analysis rather than biochemical alteration of DNA-DNA interactions in intact cells. Appropriate stringency conditions should therefore be determined by experiment rather than calculated theoretically for gene evaluation in cells and tissues.