Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of SERCA2a, the cardiomyocyte sarcoplasmic reticulum Ca²⁺ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA), or by overexpression of the novel protein kinase C (PKC) isoenzymes PKC and PKC. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate using an RSV -galactosidase reporter system that a 609 nt fragment of the SERCA2a mRNA 3' UTR, containing 5 AU-rich regions, may be responsible for destabilizing the message following PMA treatment. UV-crosslinking analysis demonstrated that several proteins found in NRVM cell extracts bind to the 609nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3' UTR mRNA pull-down assays and Western blotting indicated that the AU binding protein AUF1 interacted with the SERCA2a 3' UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the post-transcriptional regulation of the SERCA2a message in NRVM.