ATP-binding cassette (ABC) proteins are one of the largest protein families found in all organisms. More than 120 genes encoding ABC proteins have been identified in Arabidopsis and rice. Various functions of plant ABC proteins have been characterized so far, and most of these proteins contain transmembrane domains. However, at present there has been no report on one soluble ABC protein group, the ABCF subfamily. We analyzed expression of five members of the Arabidopsis ABCF subfamily using promoter-GUS fusion constructs, and identified that these members are expressed in various organs of Arabidopsis at different stages. We also isolated knockout lines of four members of the ABCF subfamily, and showed that AtABCF3 is involved in root growth and development. These results suggest that the Arabidopsis ABCF subfamily functions throughout the plant and that it has important roles in development.