Human arrest defective 1(hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, and to detect the hARD1 protein in tumor tissues in vivo, we cloned the hARD1 gene from Hela cell and constructed the expression plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21 (DE3) plysS. The hARD1 protein was expressed by inducing with IPTG (1 mM) and was purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen to immunize the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer, and lung cancer. Especially, the hARD1 expression frequency in breast cancer was up to 70%, which is higher than that in the other tumors. These results indicate that the high expression level of hARD1 can be an indicator of breast cancer. This new finding will be a foundation to further explore the relationship between breast tumor and hARD1. © 2008 Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology.