TCL/RhoJ is a Cdc42-related Rho GTPase with reported activities in endothelial cell biology and angiogenesis, metastatic melanoma, and corneal epithelial cells; however, less is known about how it is inherently regulated in comparison to its closest homologues TC10 and Cdc42. TCL has an N-terminal extension of 18 amino acids in comparison to Cdc42, but the function of this amino acid sequence has not been elucidated. A truncation mutant lacking the N terminus (N) was found to alter TCL plasma membrane localization and nucleotide binding, and additional truncation and point mutants mapped the alterations of TCL biochemistry to amino acids 17-20. Interestingly, whereas the TCL N mutant clearly influenced nucleotide exchange, deletion of the N terminus from its closest homologue, TC10, did not have a similar effect. Chimeras of TCL and TC10 revealed amino acids 121-129 of TCL contributed to the differences in nucleotide loading. Together, these results identify amino acids within the N terminus and a loop region distal to the nucleotide binding pocket of TCL capable of allosterically regulating nucleotide exchange and thus influence membrane association of the protein.