Endo-beta -N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has a high level of transglycosylation activity. To determine which amino acids are involved in this activity, we employed deletion analysis, as well as random and site-directed mutagenesis. Using PCR random mutagenesis, 11 mutants with greatly decreased levels of enzyme activity were isolated. Six catalytically essential amino acids were identified by site-directed mutagenesis. Mutants E173G, E175Q, D206G, and D270N had markedly reduced hydrolysis activity, while mutants V109D, E173D, and E173Q lost all enzymatic activity, indicating that Val-109 and Glu-173 are important for the catalytic function. Moreover, we isolated a random mutation that abolished the transglycosylation activity without affecting the hydrolysis activity. The Trp-216 to Arg mutation was identified, by site-directed mutagenesis, as that responsible for the loss of transglycosylation activity. While other mutants of Trp-216 showed reduced activity, mutation to another positively charged residue (Lys) also abolished the transglycosylation activity. Sequence comparison with two other endo-beta -N-acetylglucosaminidases, that possess transglycosylation activity and that have been cloned recently, reveals a high degree of identity in the N-terminal regions of the three enzymes. These results indicate that the tryptophan residue at position 216 of Endo-A has a key role in the transglycosylation.