Two mammalian phospholipase D (PLD) isozymes (PLD1 and PLD2) have been reported. In this study, we differentially tagged these isozymes with enhanced green fluorescent protein (EGFP-rPLD1 and EGFP-rPLD2) or Xpress peptide epitope (Xpress-rPLD1 and Xpress-rPLD2) to examine the association between these isozymes. Overexpressed EGFP-rPLD1 coimmunoprecipitated with Xpress-rPLD1 using anti-Xpress antibody. However, the coimmunoprecipitation was independent of the activity of rPLD1. Xpress-rPLD2 also bound to EGFP-rPLD1 although the binding was less efficient than observed with Xpress-rPLD1. The association between rPLD2 and rPLD1 was confirmed by coimmunoprecipitation of EGFP-rPLD2 with Xpress-rPLD1. EGFP-rPLD2 also bound to Xpress-rPLD2 as shown by coimmunoprecipitation. Immunofluorescence staining of COS-7 cells coexpressing EGFP-rPLDs and Xpress-rPLDs showed that the PLD isozymes colocalized in the perinuclear and plasma membrane regions, suggesting that they could associate in a cellular setting. These results suggest that rPLD1 and rPLD2 can exist as homodimers and can form heterodimers.