We have been studying the role of the evolutionarily conserved prohibitin gene in cellular immortalization and tumor suppression. Immortalized human cells are classified into four complementation groups (A, B, C, and D) based on the ability of fusion hybrids to become senescent. The present study expands our preliminary evidence showing that the antiproliferative activity of prohibitin is only effective in immortalized Group B cells and normal cells. Data presented here show that the expression of a prohibitin mRNA with a long 3' untranslated region (3'UTR) and prohibitin protein is elevated in immortalized cells from all complementation groups. However, all immortalized cells classified in complementation Group B, and no cell lines in any of the other groups, are sensitive to the antiproliferative activity of wild-type prohibitin transcripts. All Group B cells are also homozygous for one of two human prohibitin alleles that are distinguishable by two distinct intron polymorphism restriction sites. Interestingly, sequence analysis of the prohibitin gene from representatives of each of the complementation groups showed that the 3'UTR from Groups A, C, and D matched wild type; however, the sequence from all four Group B cell lines differed from wild type. Functional inhibition assays on truncated wild-type mRNA transcripts as well as 3'UTR specific wild-type and mutated transcripts show that the antiproliferative activity of prohibitin resides, at least in part, in the 3'UTR. These data suggest that the prohibitin 3'UTR may function as a trans-acting regulatory RNA (riboregulator) whose tumor suppressor activity has been inactivated by mutation in Group B cells.