Homeodomain proteins play important roles in animal development by controlling the expression of genes involved in determining cell fates. The recently cloned human Cart1 gene encodes a paired-class homeodomain (hCART1), whose rodent homolog is mainly expressed in early chondrocytes and in prechondrocytic mesenchymal cells. To better understand its role as a transcription factor, the author has selected specific hCART1 binding sites from a random pool of oligonucleotides. It is reported here that all sites obtained contain a palindrome consisting of two TAAT sequences separated by three or four base pairs. In electromobility shift assays, recombinant hCART1 proteins bind to a palindromic probe as a multimer, possibly a homodimer. In transient transfection assays, hCART1 activates transcription from reporter plasmids containing hCART1 binding sites in HeLa cells, demonstrating both site-dependence and dosage-dependence. It is also shown here that hCART1 localizes to nucleus. These data indicate that hCART1 is a sequence-specific transcription activator in HeLa cells. In combination with data from previous studies in which hCART1 represses transcription in different cell types and promoters, they suggest that hCART1 may be a transcription modulator with both repression and activation activities.