We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D₃, a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER−/PR−/HER-2− phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present. [Cancer Res 2007;67(14):6574–81]