Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes both transamidation and GTPase reactions. In cell culture models tTG-mediated transamidation positively regulates many processes that occur in vivo during the mammalian brain growth spurt (BGS), including neuronal differentiation, neurite outgrowth, synaptogenesis and cell death mechanisms. However, little is known about the levels of tTG expression and transglutaminase (TG) activity during mammalian brain development. In this study, C57BL/6 mouse forebrains were collected at embryonic day (E) 12, E14, E17, postnatal day (P) 0, P7 and P56 and analyzed for tTG expression and TG activity. RT-PCR analysis demonstrated that tTG mRNA content increases during mouse forebrain development, whereas immunoblot analysis demonstrated that tTG protein content decreases during this time. TG activity was low in prenatal mouse forebrain but increased fivefold to peak at P0, which corresponds with the beginning of the mouse BGS. Further analysis demonstrated that the lack of temporal correlation between tTG protein content and TG activity is the result of an endogenous inhibitor of tTG that is present in prenatal but not postnatal mouse forebrain. These results demonstrate for the first time that tTG enzymatic activity in the mammalian forebrain is developmentally regulated by post-translational mechanisms.