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Simple Summary: Cancer cells from patients who carry a disease-causing BRCA1 or BRCA2 mutation are incapable of repairing double-strand DNA breaks via the homologous recombination (HR) repair pathway. As a consequence, these patients respond to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). However, using a functional read-out for HR, we show that not all HR-deficient cell lines carry a BRCA1/2 mutation. Moreover, the majority of BRCA1/2-mutant cell lines are HR proficient, highlighting the importance of measuring the functional HR status. For almost all of the BRCA1/2-mutant HR proficient cell lines, we could explain the discrepant HR status. The observed reversion mechanisms predominantly acted on restoring the BRCA1 or BRCA2 mutation directly. Taken together, our results indicate that only determining BRCA1/2 mutation status may not suffice for selecting patients for platinum-based chemotherapy or PARPi. Moreover, for experimental approaches cell line functionality of HR should be assessed beforehand. Tumors with a pathogenic BRCA1/2 mutation are homologous recombination (HR)-deficient (HRD) and consequently sensitive to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). We hypothesized that functional HR status better reflects real-time HR status than BRCA1/2 mutation status. Therefore, we determined the functional HR status of 53 breast cancer (BC) and 38 ovarian cancer (OC) cell lines by measuring the formation of RAD51 foci after irradiation. Discrepancies between functional HR and BRCA1/2 mutation status were investigated using exome sequencing, methylation and gene expression data from 50 HR-related genes. A pathogenic BRCA1/2 mutation was found in 10/53 (18.9%) of BC and 7/38 (18.4%) of OC cell lines. Among BRCA1/2-mutant cell lines, 14/17 (82.4%) were HR-proficient (HRP), while 1/74 (1.4%) wild-type cell lines was HRD. For most (80%) cell lines, we explained the discrepancy between functional HR and BRCA1/2 mutation status. Importantly, 12/14 (85.7%) BRCA1/2-mutant HRP cell lines were explained by mechanisms directly acting on BRCA1/2. Finally, functional HR status was strongly associated with COSMIC single base substitution signature 3, but not BRCA1/2 mutation status. Thus, the majority of BRCA1/2-mutant cell lines do not represent a suitable model for HRD. Moreover, exclusively determining BRCA1/2 mutation status may not suffice for platinum-based chemotherapy or PARPi patient selection. [ABSTRACT FROM AUTHOR]