Satellite DNA sequences have been studied in several groups of organisms. However, until now this type of sequence has not been characterized in cyclostomata, an evolutionarily important class of vertebrates. In the present work, we report the molecular characterization of a new family of satellite DNA in lampreys (Petromyzon marinus). Digestion of lamprey DNA with EcoRI identified a series of very abundant AT-rich (60% A+T) repeating units, with short stretches of AT, that are multimers of 370 bp. Southern blot analysis and comparison with the satellite DNA sequences deposited in the databases indicate that this new family of satellite DNA is exclusive to lampreys. The distribution of this EcoRI satellite DNA on lamprey chromosomes was analyzed by in situ hybridization. The evolutionary origin of this satellite is briefly discussed.