The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.