The importance of intronic sequences for expression of the mouse ribosomal protein gene rpL32 was evaluated by transfection experiments with a series of mutant constructs in which one or more of the three rpL32 introns was totally or partially deleted. When transiently transfected into monkey kidney (COS) cells or stably transfected into mouse L cells, a mutant that lacked all three introns was completely inactive. Constructs that contained intron 1, either alone or in combination with another intron, were expressed as efficiently as was the normal intact rpL32 gene. Constructs that lacked intron 1 but contained another spliceable intron, even one from a foreign gene, were expressed at about 10 to 20% of the maximum level. These results indicated that intron 1 contains an element that increases the level of expression by 5- to 10-fold. A comparison of internal deletion mutants localized the element to within the first 27 base pairs of intron 1. Nuclear run-on experiments with stably transfected COS cells demonstrated that this element functions at the transcriptional level. The element was inactive when translocated to a position upstream of the transcriptional start site or to a position within intron 3, which indicated that it does not have the properties of a typical enhancer. From these and other results, we conclude that introns have both a general and a specific role in rpL32 expression. The general role, which can be satisfied by any spliceable intron, is to ensure an efficient yield of RNA transcripts. The specific role is uniquely attributable to intron 1, which contains a transcriptional regulatory element near its 5' end.