The gene (hprt) coding for mouse HPRT (hypoxanthine phosphoribosyltransferase) is transcribed from a promoter lacking CAAT and TATAA boxes. It is expressed ubiquitously, albeit at different levels, in all tissues and cultured cells. During investigations to characterise hprt transcription control elements required in embryonic stem (ES) cells and to develop compact hprt minigenes for gene-targeting strategies, we discovered a requirement for intron-1 sequences for expression in ES cells. The essential intron-1 element, which is 420 bp long, is located 230 bp downstream from the transcription start point and is shown to increase transcription from the hprt promoter in a position- and orientation-dependent manner. We propose that this element is an integral downstream part of the hprt promoter.