Summary In vivo protein–protein interactions are frequently studied by means of yeast two‐hybrid analysis. However, interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA‐binding (BD) and activation domains (AD) we established an Arabidopsis protoplast two‐hybrid (P2H) system. The use of Gateway®‐compatible vectors enables the high‐throughput screening of protein–protein interactions in plant cells. The efficiency of the system was tested by examining the homo‐ and heterodimerization properties of basic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsis thaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast and protoplast two‐hybrid experiments. Surprisingly, almost no homodimerization but rather specific and selective heterodimerization was detected. Heterodimers were preferentially formed between group C members (AtbZIP9, ‐10, ‐25, ‐63) and members of group S1 (AtbZIP1, ‐2, ‐11, ‐44, ‐53). In addition, significant but low‐affinity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitative approach, P2H identified weak heterodimerization events which were not detected in the yeast system. Thus, in addition to cell biological techniques, P2H is a valuable tool for studying protein–protein interaction in living plant cells.