Metallothioneins (MTs) are multipurpose proteins with clear antioxidant, anti-inflammatory and metal homeostasis properties. The roles of brain MT-1 and MT-2 are similar to those described in the periphery, and are inducible by metals, inflammatory and stress stimuli. MT-3, originally named growth inhibitory factor, exists mainly in the central nervous system, is hardly ever inducible and its functional role and regulation are poorly understood and controversial. In the present study we examined how absence of MT-3 affects phenotypic characteristics and its effects on MT1/2 expression in basal situation and after induction. Hyperactive behavior was found only in young male Mt-3 KO mice and disappeared in the older ones. Absence of MT-3 was associated with a significant increase of MT-1/2 protein levels in several brain areas but decreased MT-1 mRNA levels, which might be related to lower corticosterone levels. The response to stress or inflammation on corticosterone plasma levels was similar in wild type and Mt-3 KO mice, suggesting that the relevant MT-3 role as MT-1/2 regulator in basal conditions is lost when other important regulatory factors such as glucocorticoids or cytokines appear.