Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wild-type) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wild-type cells, basal bodies in centrin-RNAi cells were separated from the spindle poles, suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.