Summary: A Cre recombinase expression cassette was inserted into the X‐linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this “Cre‐deleter” transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene. genesis 32:199–202, 2002. © 2002 Wiley‐Liss, Inc.