We have examined the temporal and spatial distribution of myofibroblast-like cells, a phenotype with fibroblast and smooth muscle features, in an experimental model of renal infection. Escherichia coli organisms (105) were inoculated directly into the renal cortex of Sprague-Dawley rats weighing 270 g. Saline was substituted in a control group. The animals were sacrificed at five time points up to day 24 (E. coli n = 8, controls n = 3 each interval). Myofibroblasts were identified by morphology and immunohistochemistry for alpha smooth muscle actin (Α-SMA) and compared with staining for monocytes (ED-1), collagen III, and bromodeoxyuridine incorporation. Histological changes included a focal lesion in E. coli infected animals. Interstitial Α-SMA staining was confined to spindle-shaped cells resembling myofibroblasts. The percent fractional area of Α-SMA staining in the lesion increased from 0.12 ± 0.09 at day 1 to 20.0 ± 7.1 at day 3 (p < 0.005), decreasing progressively to 2.0 ± 2.6 by day 24. This paralleled bromodeoxyuridine incorporation in myofibroblasts: 0.4 ± 0.5 cells/0.25 mm2 at day 1, 105.0 ± 36.3 at day 3, and 2.6 ± 2.2 cells/0.25 mm2 at day 24. ED-1-positive cells increased from 374 ± 200/ 0.25 mm2 at day 1 to 894 ± 88 at day 3 (p < 0.01), declining to 230 ± 108/ 0.25 mm2 by day 24. Intracellular collagen III and Α-SMA stainings were colocalized at day 3. The fractional area of collagen III increased by day 24 (p < 0.05). In conclusion, myofibroblasts accumulate transiently during renal interstitial fibrosis and are derived at least in part from local proliferation. Double labeling suggests that myofibroblasts may be synthetically active.