We investigated the interaction of UO₂²⁺ with peptidoglycan (PG), the main part of the outer membrane of Gram-positive bacteria, by potentiometric titration and time-resolved laser-induced fluorescence spectroscopy (TRLFS) over a wide pH (2.0 to 9.0) and concentration range (10⁻⁵ to 10⁻⁴ M U(VI), 0.01 to 0.2 g L⁻¹ PG). With potentiometry two different dissociation constants for the carboxyl sites of glutamic acid and diaminopimelic acid (pK_a = 4.55 ± 0.02 and 6.31 ± 0.01), and one averaged pK_a for hydroxyl and amino groups (which are not distinguishable) (9.56 ± 0.03) and the site densities could be identified. With potentiometry three different uranyl PG complexes were ascertained: two 1 : 1 uranyl carboxyl complexes R–COO–UO₂⁺, one with the glutamic acid carboxyl group (log β₁₁₀ = 4.02 ± 0.03), which has a very small formation ratio, and one with the diaminopimelic acid carboxyl group (log β₁₁₀ = 7.28 ± 0.03), and a mixed 1 : 1 : 1 complex with additional hydroxyl or amino coordination, R–COO–UO₂⁽⁺⁾–A_i–R (A_i = NH₂ or O⁻) (log β₁₁₁₀ = 14.95 ± 0.02). With TRLFS, also three, but different, species could be identified: a 1 : 1 uranyl carboxyl complex R–COO–UO₂⁺ (log β₁₁₀ = 6.9 ± 0.2), additionally a 1 : 2 uranyl carboxyl complex (R–COO)₂–UO₂ (log β₁₂₀ = 12.1 ± 0.2), both with diaminopimelic acid carboxyl groups, and the mixed species R–COO–UO₂⁽⁺⁾–A_i–R (A_i = NH₂ or O⁻) (log β₁₁₁₀ = 14.5 ± 0.1). The results are in accordance within the errors of determination.