Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and anticancer activities. They are known to act as H+/Cl- symporters possibly through electrostatic binding to Cl- that facilitates proton-coupled transmembrane transport of halides. This activity has been ascribed to their promotion of apoptosis by acidification of the intracellular pH (pHi). Since the protonated pyrromethene chromophore of Prod (1) is expected to play a critical role in pHi regulation, and the A-pyrrole ring is known to be important for anticancer activity, we prepared several Prod analogues with various A-ring systems to determine their proton affinity in 1:1 (v/v) acetonitrile (MeCN)/H(2)O and anticancer properties against HL-60 cancer cells. Our studies show that the A-ring strongly influences the proton affinity of the pyrromethene entity. Replacement of the C-2 methoxy group in 2,4-dimethoxy-pyrromethene 3 (apparent pK(a) = 4.95) with the A-pyrrole ring to generate the Prod analogue 5 raised the apparent pK(a) to 7.54 (increase by 2.59 pK units) and caused a 76 nm red shift in the UV-vis absorbance of the protonated species (AH+). The A-pyrrole NH atom plays an important role in stabilization of AH+, as its replacement with O or S atoms decreases the apparent pK(a) by 0.79 and 1.07 pK units, respectively. A 4-substituted phenyl series of Prod analogues 8-14 exhibited a linear correlation with the Hammett sigma(p) values. Within the phenyl series, two Prod analogues were found to inhibit colony formation of HL-60 cancer cells, although the inhibition did not correlate with the proton affinity of the pyrromethene entity. The implications of these findings with regard to the anticancer activities of the prodigiosins are discussed.