The relationship of green algae to land plants has greatly interested botanists for more than a century. In recent years, several characters, particularly ultrastructural ones, have been used to define a green algal group (Charophyceae) from which land plants are thought to have arisen. Here we provide the first molecular genetic evidence in support of the charophycean origin of land plants. Group II introns have previously been found in both the tRNAAla and tRNAIle genes of all land plant chloroplast DNAs examined, whereas all algae and eubacteria examined have uninterrupted genes. The distribution of these introns in Coleochaete, Nitella and Spirogyra, members of the Charophyceae, confirms that these taxa are part of the lineage that gave rise to land plants. Furthermore, the intron data place Coleochaete and Nitella closer to land plants than Spirogyra. These introns were most probably acquired by the chloroplast genome more than 400-500 million years ago, the time of land plant origin.