In hematuria, dysmorphic red blood cells found in the urine sediment are known to indicate glomerular origin of bleeding. To better understand the mechanisms which might cause the typical membrane changes in dysmorphic erythrocytes in vivo, we have exposed normal human erythrocytes in vitro to an osmotic and enzymatic environment similar to that of the nephron. In addition, treated or untreated erythrocytes were exposed to a hemolytic environment. The morphology of the cells was evaluated by light and electron microscopy in comparison of that of dysmorphic erythrocytes obtained in vivo. Neither the passage of erythrocytes in sequence through various solutions each simulating the tubule fluid in a different part of the nephron, nor exposure to urinary or renal enzymes or to serum per se causes the typical membrane alterations. Upon exposure of passaged erythrocytes to a hemolytic environment, however, 50 to 90% of the cells become dysmorphic in a time and dose dependent fashion. By light and electron microscopy these dysmorphic erythrocytes obtained in vitro are indistinguishable from those obtained in vivo. In contrast, non-passaged red blood cells are not affected by the same hemolytic environment. We conclude that osmotically challenged erythrocytes exposed to a hemolytic environment develop dysmorphic membrane alterations.