Titin, a major contributor to cardiomyocyte (CM) stiffness, is expressed in two main isoforms in mammalian hearts, stiff N2B (3.0MDa) and compliant N2BA (>3.2MDa). The N2B:N2BA ratio changes greatly during development: embryonic rat hearts express a 3.7MDa fetal N2BA-isoform, which is subsequently replaced by shorter N2BA-titins and N2B. To determine factors regulating the titin-isoform switching, 2% SDS-PAGE was used to analyze titin expression in CM cultures prepared from E18 rats. Under standard culture conditions, the N2B-percentage increased after day 2 to reach on average 73% of total titin on day 9. Radial stretch-release cycles applied over four culture days did not alter the N2B:N2BA ratio. However, CMs cultured from day 3 on in the absence of fetal calf serum expressed relatively less N2B (65%) on day 9, suggesting a role for serum components (growth factors, hormones) in titin-isoform switching. Indeed, the N2B- percentage increased to only 51% in CMs cultured for 9 days in serum depleted of steroid hormones. Conversely, in serum-free medium supplemented with angiotensin-II or triiodo-L-thyronine (T3), N2B reached 73% and 78%, respectively; endothelin-1 did not alter N2B%. The T3-effect on titin expression was unrelated to changes in cell size or contractility and was not prevented by Bisphenol-A, a specific inhibitor of T3-receptors. We conclude that T3 is a regulator of titin expression in rat CMs. T3 likely affects titin-isoform switching via a non-genomic pathway.