Human DNA sequences in the human-hamster somatic cell hybrid XJM12.1.3 exist in the form of a minichromosome including the centromere of human chromosome 1. We describe the cloning of XJM12.1.3 DNA into the lambda vector EMBL3, the identification of minichromosome DNA-containing recombinants by hybridization with human sequences, and the characterization of one recombinant as a specific and unique probe for a region close to the centromere of human chromosome 1. This probe and others isolated from the minichromosome DNA are being developed to permit molecular access to a human centromere and its functional sequences.