Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, stress responses, protein trafficking, and membrane metabolism. This report describes the cloning and characterization of three novel PLD genes from rice, designated RPLD3, RPLD4 and RPLD5. The rice PLDs, including the previously isolated RPLD1 and RPLD2, are similar to PLD subfamilies of Arabidopsis: Based on sequence homology and domain conservation, RPLD1 is most similar to the PLDalpha subfamily of PLDs while RPLD5 most closely resembles the PLDdelta type. RPLD2, 3 and 4 represent a unique subfamily, although they are most similar to PLDalpha. RPLD1 is located on chromosome 1, RPLD5 on chromosome 3, and RPLD2, RPLD3, and RPLD4 are tandemly arrayed on chromosome 5. Transcriptional analysis reveals that RPLD1, present in healthy rice vegetative tissues, is induced rapidly but transiently in wounded leaf tissues. RPLD2, also induced by wounding, is present at lower levels but for a more prolonged duration than RPLD1. Immunolocalization with peptide specific antibodies to each of the five PLDs was used to demonstrate that the isoforms have overlapping but distinct patterns of distribution in healthy rice cells. RPLD1 was detected in mesophyll cell wall, membranes, and chloroplasts, whereas RPLD3 and RPLD4 were located predominantly in the chloroplasts. Labeling of RPLD2 and RPLD5 was sparse, and was most concentrated in the secondary walls of xylem (RPLD2) and guard cells (RPLD2 and RPLD5). This combined information on structural features, expression profiles, and cellular localization will assist the basis for dissection of PLD isoform function in rice.