FSH is an alpha:beta heterodimeric pituitary glycoprotein that shares a common alpha-subunit with LH and TSH. To study the role of FSH in mammalian reproduction, we have previously generated an FSH-deficient mouse model using embryonic stem (ES) cell technology by introducing a null mutation in the unique FSHbeta gene. Male mice deficient in FSH are fertile despite their small testes and reduced sperm number and motility. In contrast, FSH-deficient female mice are infertile due to a block in folliculogenesis at the preantral stage. In this set of experiments, we have rescued the mutant phenotypes of FSHbeta-deficient mice by two genetic strategies. In the type I rescue mice, we introduced into the FSHbeta-deficient background a 10-kb human FSHbeta transgene that is selectively expressed in pituitary gonadotropes. The presence of this transgene [and thus the interspecies hybrid (i.e. mouse alpha:human FSHbeta hormone)] in the background of mouse FSHbeta deficiency completely restored the testis size, sperm number, and motility defects to levels comparable to those seen in control male mice. All of the mouse FSHbeta-deficient female mice carrying this human FSHbeta transgene resumed normal folliculogenesis, were fertile and delivered normal size litters. In the type II rescue mice, human FSH (human alpha:human FSHbeta) was ectopically produced from multiple tissues in the mutant background using a mouse metallothionein-I promoter. Whereas ectopic production of human FSH completely rescued the mouse FSHbeta-deficient male mice, only 3 out of 10 mouse FSHbeta-deficient females bearing these human FSH transgenes were fertile and carried their pregnancies to term and parturition. We conclude that the resultant phenotypes due to a genetic deficiency of mouse FSHbeta can be corrected by appropriate expression of human FSH transgenes and that human FSHbeta gene regulation and function in the mouse pituitary are indistinguishable from the endogenous mouse FSHbeta gene.