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Titin is the largest known protein and a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Accumulating evidence that mRNA transcripts are post-transcriptionally regulated by specific motifs located in the flanking untranslated regions (UTRs) led us to consider the role of titin 5′-UTR in regulating its translational efficiency. Titin 5′-UTR is highly homologous between human, mouse, and rat, and sequence analysis revealed the presence of a stem-loop and two upstream AUG codons (uAUGs) converging on a shared in frame stop codon. We generated a mouse titin 5′-UTR luciferase reporter construct and targeted the stem-loop and each uAUG for mutation. The wild-type and mutated constructs were transfected into the cardiac HL-1 cell line and primary neonatal rat ventricular myocytes (NRVM). SV40 driven 5′-UTR luciferase activity was significantly suppressed by wild-type titin 5′-UTR (∼70% in HL-1 cells and ∼60% in NRVM). Mutating both uAUGs was found to alleviate titin 5′-UTR suppression, while eliminating the stem-loop had no effect. Treatment with various growth stimuli: pacing, PMA or neuregulin had no effect on titin 5′-UTR luciferase activity. Doxorubicin stress stimuli reduced titin 5′-UTR suppression, while H 2 O 2 had no effect. A reported single nucleotide polymorphism (SNP) rs13422986 at position -4 of the uAUG2 was introduced and found to further repress titin 5′-UTR luciferase activity. We conclude that the uAUG motifs in titin 5′-UTR serve as translational repressors in the control of titin gene expression, and that mutations/SNPs of the uAUGs or doxorubicin stress could alter titin translational efficiency. [ABSTRACT FROM AUTHOR]