We have isolated two Arabidopsis thaliana cDNA clones that encodes different H1 histone proteins. The H1-1 and H1-2 proteins are 274 and 273 amino acids in length, respectively. Unlike the H1 histones within a single animal species, the two plant H1 proteins share little sequence similarity outside the protein's central globular domain. Within the globular domain, a pentapeptide that is extremely well conserved in animal H1 histones, is not found in either of the plant proteins. Southern blot analysis suggests that A. thaliana has only three H1 histone genes. A genomic clone encoding the H1-1 protein was isolated and the protein-coding region was found to consist of two exons separated by a 104-bp intron. The site of transcriptional initiation of the H1-1 gene was mapped by primer-extension analysis and a conserved octamer motif, identical to that observed in most plant core histone genes that have been characterized to date, was found 101 nucleotides upstream of the presumed transcription-initiation site. The 3' portion of the gene encoding H1-2 was also isolated and sequenced. When the 3'-flanking regions of the two H1 genes were compared, several highly conserved sequences were observed that might be convergently transcribed relative to the histone genes.