We have shown previously that acute heat shock protein (Hsp) 70 induction in a human melanoma cell line containing a doxycycline-inducible Hsp70 expression construct increases lysability of these tumor cells by cytotoxic T lymphocyte (CTL) without interfering with MHC class I expression and antigen presentation. The same parental melanoma cell line has now been transduced retrovirally to overexpress Hsp70 permanently. Here we demonstrate that MHC class I cell surface expression is again not altered and that these cells, in contrast with acutely Hsp70 overexpressing cells, do not show augmentation of CTL-mediated apoptosis. Also, long-term induction of Hsp70 in cells with the doxycycline-inducible Hsp70 construct leads to abrogation of increased lysability. Because, furthermore, after heat shock the same permanently Hsp70 overexpressing cells show Hsp70 induction and increased lysability, it is hypothesized that acutely available Hsp70 is able to chaperone proteins that are involved in CTL-mediated apoptosis of target cells and to thereby improve their lysability. We also observed that permanent but not acute Hsp70 overexpression resulted in decreased levels of Hsc70, the constitutively expressed member of the Hsp70 family. Down-regulation of Hsc70 occurs at the post-transcriptional level and can be observed also after long-term induction of Hsp70 in cells containing the doxycycline-inducible expression system. Hsc70 down-regulation might reflect a functional integration of the overexpressed Hsp70 on the basis of a chaperone network so that only acute induction will provide Hsp70 that can improve tumor cell lysability. The implications of the differential effect of acute versus permanent Hsp70 overexpression for tumor therapy are discussed.