The protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3) regulates the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) which regulates transcription of genes involved in cell growth, proliferation and apoptosis. The conserved proline, isoleucine, asparagine, isoleucine, threonine (PINIT) domain of PIAS3 is thought to promote STAT3–PIAS3 interaction. The (His)7‐PINIT domain (PIAS385–272) was heterologously expressed and purified to homogeneity by nickel affinity and size exclusion chromatography, and shown to be a folded monomer in solution. Using surface plasmon resonance spectroscopy (SPR) the PINIT domain (PIAS385–272) alone was shown to specifically bind to STAT3 in a concentration dependent manner. L97A, R99N and R99Q mutations of the PINIT domain were found to abrogate binding to STAT3, suggesting that these residues were part of a potential binding surface. An homology model for the PINIT domain was calculated to analyse the potential locations of L97 and R99 in the structure, and to evaluate the potential role of these residues in interactions with STAT3. Copyright © 2011 John Wiley & Sons, Ltd.