The ARE/poly-(U) binding factor 1 (AUF1), a protein family consisting of four isoforms, is believed to mediate mRNA degradation by binding to AU-rich elements (ARE). However, evidence exists that individual AUF1 isoforms may stabilize ARE-containing mRNAs. The 3'-untranslated region of the human inducible nitric-oxide synthase (iNOS) contains five AREs, which promote RNA degradation. We have recently shown that the RNA-binding protein KSRP is critically involved in the decay of the iNOS mRNA. In this study we examined the effects of the individual AUF1 isoforms on iNOS expression. Overexpression of each AUF1 isoform reduces iNOS expression on mRNA and protein levels to the same extent by modulation of mRNA stability. Accordingly, knockdown of all or individual AUF1 isoforms by an RNA interference approach enhances iNOS expression. The AUF1 effect on iNOS expression is dependent on the iNOS 3'-untranslated region sequence, as demonstrated in transfection experiments with a reporter mRNA. Binding studies showed that all AUF1 isoforms interact with the same AU-rich region in the iNOS-3'-untranslated region. Cytokine stimulation altered intracellular AUF1 binding activities. These data demonstrate that AUF1 is an important factor that promotes iNOS mRNA degradation. Furthermore, all individual AUF1 isoforms act in a similar manner.