The concentration of steroid glucuronides in serial samples of early morning urine (EMU) can be used to predict the fertile period in the female menstrual cycle. The monoclonal antibody 4155 has been used as a convenient means of measuring the concentration of steroid glucuronides in EMU, as it specifically recognises the steroid hormone estrone beta-D-glucuronide (E3G), with very high affinity, and the closely related hormone estriol 3-(beta-d-glucuronide) (EI3G), with reduced affinity. Although 4115 binds these hormones with different affinities, EI3G differs from E3G only in the addition of a hydroxyl group and reduction of an adjacent carbonyl. To investigate the structural basis of this fine binding specificity, we have determined the crystal structures of the variable fragment (Fv) of 4155 in complex with each of these hormones.Two crystal forms of the Fv4155-EI3G complex, at resolutions of 2.1 A and 2.5 A, and one form of the Fv4155-E3G complex, at 2.1 A resolution were solved and refined. The crystal structures show the E3G or EI3G antigen lying in an extended cleft, running form the centre of the antibody combining site down one side of the variable domain interface, and formed almost entirely from residues in the heavy chain. The binding cleft lies primarily between the heavy chain complementarity determining regions (CDRs), rather than in the interface between the heavy and light chains. In both complexes the binding of the glucuronic sugar, and rings A and B of the steroid, is specified by the shape of the narrow cleft. Analysis of the Fv structure reveals that five of the six CDR regions can be assigned to one of the predefined canonical structural classes.The difference in the binding affinity of Fv4155 for the two steroid hormones is accounted for by a subtle combination of a less favoured hydrogen-bond geometry, and a minor rearrangement of the water molecule network around the binding site. The rearrangement of water molecules results from the burial of the additional hydroxyl group of the EI3G in a hydrophobic environment.