We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.