In the present study, it was found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R2 protein expression did not correlate with mRNA expression in melanoma cell lines. In particular, early passage primary cultures from patients had low TRAIL-R2 protein expression compared with later passage cultures although TRAIL-R2 mRNA expression was similar in early and late passages. Similarly, cell lines made resistant to TRAIL by cultures in TRAIL had low TRAIL-R2 protein expression but normal levels of mRNA for TRAIL-R2. Expression from a luciferase reporter gene construct with the 3'-untranslated region (UTR) (but not the 5'-UTR) of TRAIL-R2 was suppressed when transfected into the TRAIL-selected (resistant) melanoma lines compared with that seen in the parental (sensitive) lines. Similar results were seen in early passage (resistant) cultures compared with late passage (sensitive) primary melanoma cultures. RNA gel shift assays demonstrated protein(s) binding to the 3'-UTR of TRAIL-R2 mRNA that were more evident in TRAIL-resistant cultures with low TRAIL-R2 protein expression. A 23-base fragment of the 3'-UTR inhibited binding of the proteins to the 3'-UTR, and a probe using this fragment bound to proteins in TRAIL-selected melanoma lines and early passage isolates of melanoma. Binding of the 3'-UTR probe to the cytosolic protein(s) was induced by exposure to TRAIL and was lost from the TRAIL selected lines 2-3 days after withdrawal of TRAIL from the cultures. These results are consistent with post-transcriptional regulation of TRAIL-R2 expression by cytosolic proteins induced by TRAIL that bind to the 3'-UTR region of TRAIL-R2 mRNA.