Background: The cardiomyocyte cell membrane is composed of the surface membrane and a complex system of membrane invaginations, the t-tubules. In keeping with its role in synchronisation of electromechanical coupling, the t-tubular system is distinguished from the cell membrane by its ion channel composition. Variability of the architecture of the t-tubular system might modulate electrophysiological function at the cellular level. Light and electron microscopy-based methods were established to generate three-dimensional models of t-tubular structure. Effects of t-tubular variability on electrophysiological function were investigated using a pseudo-two dimensional model of the ventricular cardiomyocyte. Methods: Confocal microscopy and electron tomography were used to generate three-dimensional models. Based on the distribution of the t- tubular membrane, electrophysiological parameters were calculated using a mathematical model. Results and Discussion: Three-dimensional models of t-tubular structure could be generated using different staining protocols for confocal microscopy and different fixation protocols for electron tomography. Advantages and applications of the different methods were discussed. The t-tubular fraction varied considerably among different cardiomyocytes. In the model this gave rise into changes in ionic currents; however variation of t-tubular density lead to heterogeous changes in repolarisation only after simulated inhibition of repolarisation-related membrane currents. Conclusions: Electron tomography allows generation of structural models at resolution of a few nanometres, however optimisation of fixation protocols is a critical step. Larger volumes can be acquired in living cells with confocal microscopy; however, a large fraction of the t-tubular system is sub-resolution. The variability of the t-tubular system may contribute to electrophysiological heterogeneity in patho-physiologically challenged states, and it forms an interesting target for functional modelling based on three-dimensional models of t-tubular structure.