The Frank-Starling response (preload-dependent modulation of contractility) can be observed in intact isolated cardiomyocytes, but little is known about regional differences in this behaviour. Here, we use a modified version [1] of the carbon fibre (CF) technique [2] to characterize mechanical parameters of cells, isolated from different regions of the Guinea pig heart, during different preloaded conditions. After Schedule 1 killing (according to UK Home Office guidance), hearts were extracted from female Guinea pigs and Langendorff-perfused with normal Tyrode solution. The tissue was enzymatically digested as described before [1], and cells were harvested separately from the atrium (A), left ventricle (LV) and right ventricle (RV). For experiments, cells were placed in a temperature controlled (37^oC) laminar flow chamber on an inverted microscope stage and stimulated at 2 Hz. CF were attached to cell ends, cells lifted off the coverslip, and CF distance was monitored by online video microscopy (IonOptix; [1]). To increase preload, cells were stretched by up to 30% of slack length (L0). Active and passive force were calculated from observed bending of calibrated CF. End-systolic and end-diastolic lengths and forces (EDL, ESL, EDF, ESF; respectively), amplitude of shortening (dL), maximal velocities of shortening (Vmax_C) and relaxation (Vmax_R), time-to-peak contraction (TTP), and time to 30% relaxation (T30) were identified for each single twitch. To correct for different cell dimensions, cross-sectional area was used to normalize force-related data, while length-related changes were additionally normalized to L0. The normalized data were used to plot x/y parameter relations, and slopes were approximated by linear regression analysis. Slopes were statistically compared for cells from different regions using ANOVA; all data presented as mean±SEM (p<0.05). Changes in EDL had more pronounced effects on dL in A myocytes (10.4±1.16μm/μm) compared to RV (8.7±0.62μm/μm) and LV (9.3±0.41μm/μm). At the same time, EDF rose nearly twice as fast with EDL in A (54.8±1.5mN/mm2) cells, compared to RV and LV (both < 30mN/mm2). The stretch induced gain in Vmax_C and Vmax_R was also significantly higher in A than in LV and RV. At the same time, TTP of A (63.3±9.37ms) cells was significantly lower than in RV (98.3±9.2ms) or LV (99.9±6.7ms). Myocytes from A had significant smaller cross sectional areas and L0 compared to RV and LV. In summary, cardiomyocytes from A are smaller, but show more pronounced contractile responses to increased EDL. This may be of functional relevance for contractile performance of the relatively thin-walled atrium.