Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σFpvI and σPvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σFpvI and σPvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR20 and the σFpvI and σPvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σFpvI and σPvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR20) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6-σFpvI/FpvR1-67 and His6-σPvdS/FpvR1-67 complexes. A comparison of these values with those previously reported for the His6-σFpvI/FpvR1-89 and His6-σPvdS/FpvR1-89 complexes is made.