Argininosuccinate lyase (ASL)/delta-crystallin, a major soluble protein of the transparent eye lens of birds and reptiles, is a mixture of tetramers comprising all possible combinations of two similar polypeptides (delta 1 and delta 2). Only the delta 2 polypeptide has ASL activity. In the present investigation we have purified each of the 5 major isoforms (delta A to delta E, pI 5.2 to 5.8) of delta-crystallin tetramers from the embryonic duck lens by isoelectric focussing and established by peptide sequencing that the delta 1 and delta 2 polypeptides are encoded in the previously identified, linked delta 1 and delta 2 genes, respectively. The relative amounts of the different tetramers in the 14-day-old embryonic lens were consistent with equal expression of the 2 delta-crystallin genes and no preference for assembly of the 2 delta polypeptides. The relative amount of ASL activity of the tetramers was a linear function of the relative amount of their delta 2 polypeptides, with delta A (only delta 1) lacking enzymatic activity altogether. delta B (3 delta 1:1 delta 2), delta C (2 delta 1:2 delta 2), delta D (1 delta 1:3 delta 2) and delta E (4 delta 2) all gave normal Michaelis-Menten kinetics for fumarate production from argininosuccinate at 40 degrees C and had a similar Km (average Km for mixture was 0.15 mM). delta E had a Km of 0.187 mM and a Vmax of 9 mumol/min per mg protein. Unlike bovine and like human ASL, both reported previously, embryonic duck ASL/delta-crystallin showed no evidence of cooperativity or activation by GTP. Each isoform had a similar far ultraviolet circular dichroism spectrum and thermal stability between 20 degrees C and 60 degrees C, with denaturation occurring at 65 degrees C. Our data suggest that gene duplication, structural modifications leading to greater thermal stability of the delta 1 and delta 2 polypeptides, and selective loss of ASL activity in the delta 1 polypeptide all occurred during the recruitment of ASL for a refractive role in the duck lens, resulting in the generation of ASL isoenzymes.