Objective: The hypothesis that elevated systolic stress induces myocyte thickening has been difficult to test directly. We tested this hypothesis in working rat right ventricular papillary muscles using a recently developed technique for long-term muscle culture. Methods: Muscles were cultured for 36 hours either isometrically at different levels of systolic stress or at physiologic amounts and rates of shortening. Results: Isometric contraction induced rapid increases in myocyte diameter regardless of the level of systolic stress, while control myocyte dimensions were maintained if physiologic amounts and rates of systolic shortening were imposed. Myocyte thickening was accompanied by a significant decrease in cell length and number of sarcomeres in series along the long axis of the myocyte, suggesting that thickening may have occurred in part by rearrangement of existing sarcomeres. Conclusions: We conclude that the pattern of systolic shortening and/or diastolic lengthening regulates myocyte shape in working rat right ventricular papillary muscles, while systolic stress plays little or no role.