We have recently isolated and characterized a homolog of the Arabidopsis thaliana IAA amidohydrolase ILR1 from Arabidopsis suecica (sILR1). This study examines the enzymatic characteristics of sILR1, as well as spatial and temporal expression of sILR1 compared to ILR1. The sILR1 protein can utilize IAA-alanine and IAA-glycine as substrates more effectively than ILR1. In contrast to ILR1, sILR1 cannot cleave IAA-phenylalanine or IAA-leucine as substrates. ILR1 and sILR1 share a pH optimum of 8.0 in Tris buffer. Based on the calculated K-max value, sILR1 has a higher affinity for IAA-alanine than ILR1. The sILR1 transcript is first detectable in seedlings at day 4 after germination and rises to a steady state level from day 5 to day 15. In A. thaliana, expression of ILR1 begins with a burst at day 1 and decreases over 15 days to a relatively low, but steady state level. Examination of ILR1 and sILR1 transcripts in different tissues shows that both sILR1 and ILR1 are highly expressed in roots, although ILR1 appears more highly expressed in hypocotyls, flowers, and basal leaves than sILR1.