Nickel is the most common inducer of contact sensitivity in humans. We previously found that overrepresentation of the TCRBV17 element in Ni-induced CD4+ T cell lines of Ni-allergic patients relates to the severity of the disease. Amino acid sequences of these beta-chains suggested hypothetical contact points for Ni2+ ions in complementarity-determining region (CDR) 1 and CDR3. To specifically address the molecular requirements for Ni recognition by TCR, human TCR alpha- and beta-chains of VB17+ Ni-reactive T cell clones were functionally expressed together with the human CD4 coreceptor in a mouse T cell hybridoma. Loss of CD4 revealed complete CD4 independence for one of the TCR studied. Putative TCR/Ni contact points were tested by pairing of TCR chains from different clones, also with different specificity. TCRBV17 chains with different J regions, but similar CDR3 regions, could be functionally exchanged. Larger differences in the CDR3 region were not tolerated. Specific combinations of alpha- and beta-chains were required, excluding a superantigen-like activation by Ni. Mutation of amino acids in CDR1 of TCRBV17 did not affect Ag recognition, superantigen activation, or HLA restriction. In contrast, mutation of Arg95 or Asp96, conserved in many CDR3B sequences of Ni-specific, VB17+ TCR, abrogated Ni recognition. These results define specific amino acids in the CDR3B region of a VB17+ TCR to be crucial for human nickel recognition. CD4 independence implies a high affinity of such receptor types for the Ni/MHC complex. This may point to a dominant role of T cells bearing such receptors in the pathology of contact dermatitis.