We investigated the role of the two calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) in the locomotor activity and motorcoordination using null-mutant mice for PV (PV-/-), CB (CB-/-) or both proteins (PV-/-CB-/-). These proteins are expressed in distinct, mainly non-overlapping populations of neurons of the central and peripheral nervous system and PV additionally in fast-twitch muscles. In a test measuring repeated locomotor activity during 18-20 days, the analysis revealed a slightly increased activity in truce lacking either protein, while the lack of both decreased the number of beams crossed during active periods. An increase in the characteristic speed during the first 8 days could be attributed to PV-deficiency, while the elimination of CB in CB-/- and double-KO mice decreased the percentage of fast movements at all time points. In the latter, additionally a reduction of the fastest speed was observed. The alterations in locomotor activity (fast movements, fastest speed) strongly correlate with the impairment in locomotor coordination in mice deficient for CB evidenced in the runway assay and the rotarod assay. The graded locomotor phenotype (CB > PV) is qualitatively correlated with alterations in Purkinje cell firing reported previously in these mice. The presence or absence of either protein did not affect the spontaneous locomotor activity when animals were placed in a novel environment and tested only once for 30 min. In summary, the lack of these calcium-binding proteins yields characteristic, yet distinct phenotypes with respect to locomotor activity and coordination.