A sequence closely linked to the Saccharomyces cerevisiae HO gene confers autonomous replication in yeast. I have subjected this putative replication origin to deletion and point mutagenesis in order to identify structural features that are important requirements for autonomous replication in vivo. This analysis identifies a 14 bp core region, which is crucial for function and shows partial sequence conservation between a number of autonomously replicating sequences. Point mutations within the core region can abolish autonomous replication. The core region is flanked on one side by a sequence of about 20 bp, which is important for efficient autonomous replication. Deletion of this flanking sequence reduces, but does not necessarily eliminate, autonomous replication.