Sex in birds is chomosomally based (ZZ male, ZW female), but the mechanism underlying sex determination remains unknown. An unresolved question is whether Z gene dosage plays a role in avian sex determination. DMRT1 is an avian Z-linked gene that shows higher expression in male gonads during embryogenesis and has been proposed as a putative testis-determining gene in birds. The Z-linkage of this gene makes it an ideal candidate for testing the question of gene dosage in avian testis determination. A higher level of DMRT1 expression in male (ZZ) versus female (ZW) embryonic gonads may reflect the presence of two Z-linked copies in the male, or it may be due to specific and active upregulation of DMRT1 during testis formation. A functional interventionist strategy was used to distinguish between these two possibilities. DMRT1 expression was analyzed in chicken embryos during experimentally induced female-to-male sex reversal, using the aromatase enzyme inhibitor fadrozole. DMRT1 expression was analyzed by whole mount in situ hybridization and reverse transcription polymerase chain reaction (for mRNA) and indirect immunofluorescence (for protein). Female-to-male sex-reversed embryos (genetically ZW) showed elevated levels of DMRT1 expression similar to those of normal males (with two copies of the Z chromosome). Elevated levels of DMRT1 are therefore associated with testis development, both in normal males (ZZ) and in sex-reversed females (ZW). SOX9 expression was also activated during female-to-male sex reversal but appeared delayed relative to DMRT1 upregulation. These results show that testis development does not require two Z-linked copies of DMRT1, but it does involve active upregulation of the gene. Higher levels of DMRT1 expression during testis differentiation therefore do not simply reflect a gene dosage difference between the two sexes but imply active involvement in male development.