Enhancers stimulate transcription of several eukaryotic genes (for review see ref. 1). While some enhancers, like that of simian virus 40 (SV40), are active in a wide range of cell types, others are more cell-specific. For example, the polyoma virus (Py) enhancer is not active in undifferentiated embryonal carcinoma (EC) cells, such as F9 cells, while it is active in differentiated cells. In contrast, the SV40 enhancer is active in both undifferentiated and differentiated EC cells. One possible explanation for this difference is that the two viral enhancers interact with different positively or negatively acting transcription factors, a notion supported by in vitro experiments showing that the Py enhancer interacts with proteins that do not bind to the SV40 enhancer. Some viral and cellular enhancers, including the Py and SV40 enhancers, can be negatively regulated by the products of the E1A transcription unit of adenovirus-2. As it has been postulated that undifferentiated F9 cells contain an E1A-like activity, it is possible that the latter is responsible for the lack of activity of the Py enhancer in these cells. We show here that the E1A products do not repress a point mutant of the Py enhancer (Py ECF9.1; ref. 11 and references therein) that is active in undifferentiated F9 cells. This result is consistent with the idea that undifferentiated F9 cells contain a cellular repressor that blocks the Py enhancer and that this repressor has the same target sequence as the E1A proteins.