The complete 1H-nuclear magnetic resonance assignments have been made for the common mono-, di-, and trihydroxy 5β-cholanoic acids; lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, and the unsubstituted parent compound, 5β-cholanoic acid, by hetero-nuclear-correlated two-dimensional NMR. The known 13C chemical shifts of these compounds were used to make the proton resonance assignments, and consistency of the carbon and proton assignments was verified by expected changes due to substituent effects. This has led to clarification of previously published 13C NMR resonance assignments. Addition of the 3α, 7α, and 12α hydroxyl substituent effects derived from the mono- and dihydroxycholanoic acids yielded predicted values for proton chemical shifts of the trihydroxy-substituted 5β-cholanoic acid, cholic acid, that agreed well with experimental values. It is suggested that the individual substituent effects can be used to predict proton chemical shifts for hydroxycholanic acids containing other combinations of 3α, 7α, 7β, and 12α hydroxyl groups.