Neonatal ventricular myocytes (CM) have long been used as an in vitro model for hypertrophy studies. In conventional 2D culture, CM lack axial orientation and rhythmic electrical stimulation. Micropatterned cultures can restrict cell attachment to narrow stripes, leading to enhanced axial orientation, particularly in spontaneously contracting CM (Rohr et. al., Circ Res, 1991). In this study, we investigated the effect of continous electrical field stimulation (CES) on micropatterned CM and examined their response to hypertrophic stimulation. Rat CM plated in serum-containing media selectively attached to stripes (100 μm x 10 mm) of fibronectin (FN) that were microcontact-printed onto coverslips. CM cultures were subjected to CES (1 Hz, 5 V/cm) for 48 hrs, with the current applied parallel or perpendicular to FN stripes. To induce a hypertrophic response, micropatterned CM were incubated for 48 hrs in serum-free medium with the α1 adrenoceptor agonist phenylephrine (PE, together with timolol). We determined that the size, minor/major axis ratio and angles relative to FN stripes of DAPI-stained nuclei can be used as surrogate measures of CM size, elongation and alignment, respectively. Compared to unpaced CM, parallel CES increased nuclear size (1028±121 vs. 798±87 μm2, P<0.001), elongation (minor/major axis: 0.76±0.10 vs. 0.84±0.08, P<0.001) and alignment (P<0.001, Mardia-Watson-Wheeler circular statistics). Perpendicular CES caused similar but significantly less pronounced changes. PE stimulation increased nuclear size (809±93 vs. 682±99 μm2, P<0.05), but did not increase elongation or alignment with or without CES. In conclusion, CES can be used to enhance the degree of differentiation of micropatterned CM due to continuous electrical activation and/or contractions and does not interfere with their hypertrophic response. Continuously paced micropatterned CM represent an advanced model for the investigation of hypertrophic responses and mechanisms and may be suitable for other applications.