The AT hook is an AT-rich DNA-binding domain that occurs three times in mammalian high-mobility-group I/Y chromosomal proteins and has recently also been identified in DNA-binding proteins from plants. We unexpectedly isolated three rice cDNA clones encoding AT hook-containing proteins in an attempt to isolate homeobox cDNA clones by south-western screening of an expression library with known binding sites for Arabidopsis and animal homeodomain proteins. One of these clones (Os-PF1) has previously been identified due to the binding of its encoded protein to PE1, a cis-acting element from the oat phytochrome promoter. The other two clones represent newly described cDNA clones, designated Os-AT1 and Os-AT2. The Os-AT1 and Os-AT2 proteins were found to have the same specificities as Os-PF1 with respect to in vitro binding of wild-type and mutant PE1 versions. However, all three proteins appeared to bind much stronger in south-western assays to two of the rather AT-rich sequences used in our screening than to the PE1 element. In none of the AT hook proteins clear homologies to transcriptional activation domains could be identified, but the N-terminal regions of Os-AT1 and Os-PF1 were found to show similarity to histone H1 chromosomal proteins. Given their structural characteristics it is conceivable that the rice AT hook proteins bind to gene promoter regions as accessory proteins that may alter the accessibility of chromatin to other nuclear factors. Their predominant expression in young and meristematic tissues suggests that the presence of the AT hook proteins may affect the expression of genes that determine the differentiation status of cells.