Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days). However, betaG-PCK-1 mRNA, which contains the entire 3'-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the betaG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3'-end of the 3'-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric betaG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the betaG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The betaG-PCK-3 mRNA that contains the 3'-half of 3'-UTR was degraded with the same half-life. Surprisingly, the betaG-PCK-2 mRNA, containing the 5'-end of the 3'-UTR, was also degraded rapidly (t((1/2)) = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3'-UTR that participate in the rapid turnover of the PEPCK mRNA.