We have developed a ligand-mimetic antibody Fab fragment specific for Drosophila PS2PS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the ECM protein Tiggrin into the H-CDR3 of the v3 ligand-mimetic antibody WOW-1. The specificity of PS2PS binding to TWOW-1 is demonstrated by numerous tests used for other integrin-ligand interactions. Binding is decreased in the presence of EDTA or RGD peptides, and by mutation of the TWOW-1 RGD sequence or the PS MIDAS motif. TWOW-1 binding is increased by mutations in the PS2 membrane-proximal cytoplasmic GFFNR sequence, or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ can be increased further by the PS2 GFFNR > GFANA mutation. A mutation in the PS I domain (PS-b58; V409>D) greatly increases ligand binding affinity, explaining the increased cell spreading mediated by PS2PS-b58. Further mutagenesis of this residue suggests that V409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin subunit PSI and stalk domains have been shown to have activating properties. We find that complete deletion of the PS PSI domain enhances TWOW-1 binding. Moreover, the PSI domain is dispensable for at least some other integrin functions, as PS-PSIdel displays an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.