Human cystatin C is a low molecular weight protein involved in the control of human cysteine proteinase activity as well as microbial cysteine proteinase activity threatening the integrity of tissues. The gene for cystatin C is located on the short arm of chromosome 20, spans 6.5 kb and has three exons. To understand the mechanisms for the expression of cystatin C at the transcriptional level we mapped the 5′ boundary of mRNA transcripts and studied the 5′-region of the cystatin C gene in a transient expression system with chimeric constructs utilizing various fragments of 1.1 kb of the 5′-flanking region coupled to the gene for human growth hormone. Mapping of the 5′-end of human cystatin C mRNA from placenta and seminal vesicles (low to medium versus high cystatin C expression, respectively) identified three major transcription initiation sites (positions -75, -78 and -80, A of initiation ATG as +1) and three minor sites (positions -98, -101 and -103). The relative amounts of different mRNA species were approximately the same in these two tissues. Functional analysis of the 5′-region in cultured HeLa cells revealed one region (positions -279 to -156) with a strong positive effect on transcription and comprising three identical tandemly arranged GC-rich sequences.