APRT (adenosine phosphoribosyltransferase) catalyzes the condensation of adenine with PRPP to form adenosine monophosphate and pyrophosphate. The enzyme is constitutively expressed in all tissues examined. The CHO APRT gene does not have a TATA or CAAT box in its 5′ region; like most housekeeping genes the 5′ region is GC-rich and contains three consensus Spl-binding sequences. A deletion of upstream region of nucleotide −89 (relative to the transcription initiation sites) does not affect gene expression, while an additional 29 base pair deletion decreases expression by two-fold. To further study the cis-acting elements essential for gene expression we constructed mutations in the 5′ region by linker-scanning techniques. We report here that (i) mutants with linker-scanning mutations between the nucleotides −33 and +22 retained 40% activity of the wild type gene; (ii) mutations in the region from nucleotides −100 to −88, and from −48 to −39 resulted in two-fold increased expression; and (iii) mutations at the transcription start sites did not affect gene expression.