Series of polycarboxybetaines (PCB-n) of pyridiniocarboxylate structure with the same degree of polymerization but differing in the number, n, of methylene groups in the alkyl spacer between charges in the betaine moieties, n = 1, 2, 3, 4, 5, and 8, were synthesized. The utility of PCB-n as positively charged components of polyelectrolyte complexes was elucidated by potentiometry, turbidimetry, and fluorescence spectroscopy. Affinity of PCB-n to the pyrenyl-tagged poly(methacrylic) acid (PMAA*) or DNA was judged from the stability of the corresponding polyelectrolyte complexes in water-salt solutions at different pH values as monitored by fluorescence quenching techniques. At pH = 9.0, PCB-1 formed the least stable complexes due to strong interaction of charged groups positioned in close proximity in the betaine moieties. The increase in n resulted in the irregular change of the affinity. Thus, as expected, PCB-2 formed noticeably more stable complexes than PCB-1. However, PCB-3 and, in particular, PCB-4 revealed weaker affinity to PMAA* or DNA that is attributed to formation of stable ion pairs between charges in the betaine rings. At neutral and slightly acidic pH values binding of all PCB-n except PCB-1 was drastically enhanced due to protonation of PCB-n carboxylic groups that occurred with a Delta pH shift of 2-3 units to higher values as compared with the protonation of free PCB-n. The ability of added polyanion to compete with the betaine carboxylic groups in binding with the pyridinium groups was supported by potentiometric titration of PCB-n mixtures with sodium poly(styrenesulfonate): for n : 2, the binding of the polyanion-competitor also shifted protonation of carboxylic groups to higher values with Delta pH of more than 2 units. Practical ramifications of the revealed role of the alkyl spacer in polyelectrolyte complexation as well as the pH-induced stabilization of the complexes that occurs under enzyme-friendly conditions might extend to areas of biotechnology, specifically in bioseparation and gene delivery.