The liver-specific expression of the senescence marker protein 2 (SMP-2) in the male rat is markedly reduced during the androgen-sensitive state of young adulthood, whereas it is up-regulated during the androgen-insensitive phases of prepuberty and senescence. Nuclear runoff studies show that the age-dependent changes in SMP-2 expression are due to transcriptional regulation of the gene. In order to explore the mechanism of the regulatory process, we have cloned the upstream flanking regions of two distinct SMP-2 genes (SMP-2A and SMP-2B) and established their nucleotide sequence. These clones contain approximately 2.2 kb of the 5'-flanking sequence, exons 1 and 2, the first intron, and a portion of the second intron. The SMP-2 genes, as well as the upstream sequences, contain the sequence motifs for a number of cis-acting regulatory elements, such as the hepatocyte-specific element (HP1) and the androgen response element (ARE). S1 nuclease and primer extension analyses have established the transcription initiation sites for these genes. For functional analysis of the upstream sequences, we have constructed a hybrid plasmid containing the SMP-2A gene sequence (-1970 to +38 bases) fused to the structural gene for chloramphenicol acetyl-transferase (CAT). Upon transfection into rat hepatoma cells (FT02B), this construct was able to drive expression of the CAT gene. The same construct, however, failed to function in fibroblast-derived L cells, indicating tissue-specific regulation of the construct promoter.