MDC1 and 53BP1 are critical components of the DNA damage response (DDR) machinery that protects genome integrity and guards against cancer, yet the tissue expression patterns and involvement of these two DDR adaptors/mediators in human tumours remain largely unknown. Here we optimized immunohistochemical analyses of human 53BP1 and MDC1 proteins in situ and identified their virtually ubiquitous expression, both in proliferating and quiescent, differentiated tissues. Focus formation by 53BP1 and/or MDC1 in human spermatogenesis and subsets of breast and lung carcinomas indicated physiological and 'pathological' activation of the DDR, respectively. Furthermore, aberrant reduction or lack of either protein in significant proportions of carcinomas supported the candidacy of 53BP1 and MDC1 for tumour suppressors. Contrary to carcinomas, almost no activation or loss of MDC1 or 53BP1 were found among testicular germ-cell tumours (TGCTs), a tumour type with unique biology and exceptionally low incidence of p53 mutations. Such concomitant presence (in carcinomas) or absence (in TGCTs) of DDR activation and DDR aberrations supports the roles of MDC1 and 53BP1 within the ATM/ATR-regulated checkpoint network which, when activated, provides an early anti-cancer barrier the pressure of which selects for DDR defects such as p53 mutations or loss of 53BP1/MDC1 during cancer progression.