In Penicillium chrysogenum, the industrial producer of the beta-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain DeltaPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain DeltaPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain DeltaPcku70, three genes related to beta-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.