Site-directed mutagenesis is of great importance for probing the structure/function relationship of proteins. Developing our previous method (Nagy et al. Anal Biochem 324:301-303, 2004), here we report a multiplex strategy for site-directed mutagenesis using PCR in one tube to introduce a single mutation into three or more genes at the same time. DNA fragments carrying the desired mutation can be distinguished from each other in a standard antibiotic selection step of the transformed bacteria. Due to this strategy the mutagenesis procedure for several genes can be accelerated.