ES mice that are derived completely from embryonic stem (ES) cells can be obtained by tetraploid embryo complementation. Many neonate ES mice die because of respiratory distress, but it is not clear what contributes to the phenomenon. Using five microsatellite DNA markers, we confirmed that our ES mice were completely derived from ES cells and contained no tetraploid component. The neonatal ES mice that exhibited respiratory distress were tested for surfactant protein B (SP-B) expression by Western blotting. These mice had no SP-B expression, and even apparently healthy adult ES mice had decreased SP-B levels and aberrant SP-B phenotypes. These data suggest that the expression of SP-B protein is an important factor in the survival of ES mice to term and adulthood.