Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.