An EGFP-h-Cre cDNA (GFP fused to a codon-optimized, "humanized" cre recombinase sequence) was inserted directly downstream of the Foxp3 ATG start codon by homologous recombination in a 188-kb C57BL/6 BAC clone (RP23-143D8) containing the entire Foxp3 gene. The purified BAC was injected into pronuclei of nonobese, diabetic mouse embryos. Several founders were generated. GFP expression and lack of functional Foxp3 protein, as indicated by inability of the transgene to rescue Foxp3 null mice were analyzed and line 1 was established. Functional cre activity was assayed by breeding mice to Gt(ROSA)26Sortm1(EYFP)Cos reporter mice, and high specificity of recombination was detected in the lymph nodes and spleen. This is the first of two lines derived from founder 1. Cre mediated recombination and GFP expression is greater than in line 1c.