Myofibroblasts play a major role in scar formation during wound healing after myocardial infarction (MI). Their origin has been thought to be interstitial cardiac fibroblasts. However, the bone marrow (BM) can be a source of myofibroblasts in a number of organs after injury. We have studied the temporal, quantitative and functional role of BM‐derived (BMD) myofibroblasts in myocardial scar formation. MI was induced by permanent coronary artery ligation in mice reconstituted with EGFP or pro‐Col1A2 transgenic BM. In the latter, luciferase and β‐galactosidase transgene expression mirrors that of the endogenous pro‐collagen 1A2 gene, which allows for functional assessment of the recruited cells. After MI, α‐SMA‐positive myofibroblasts and collagen I gradually increased in the infarct area until day 14 and remained constant afterwards. Numerous EGFP‐positive BMD cells were present during the first week post‐MI, and gradually decreased afterwards until day 28. Peak numbers of BMD myofibroblasts, co‐expressing EGFP and α‐SMA, were found on day 7 post‐MI. An average of 21% of the BMD cells in the infarct area were myofibroblasts. These cells constituted up to 24% of all myofibroblasts present. By in vivo IVIS^® imaging, BMD myofibroblasts were found to be active for collagen I production and their presence was confined to the infarct area. These results show that BMD myofibroblasts participate actively in scar formation after MI. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.