The apicomplexa parasite Toxoplasma gondii expresses two distinct proliferating cell nuclear antigens (PCNA) that exhibit distinct patterns of subcellular localization during tachyzoite growth. In all cell cycle phases, TgPCNA1 is concentrated in the nucleus, while TgPCNA2 is only concentrated in the nucleus during S-phase and uniformly distributed throughout the cell during mitosis and early G1-phase. TgPCNA1-GFP and native TgPCNA2 display a punctate staining pattern that is consistent with assembly into replication foci during S-phase; however, TgPCNA2 disassociates from replication foci before TgPCNA1-GFP. Consistent with the distinct pattern of TgPCNA2 cellular localization, homotypic TgPCNA2 interactions were primarily observed by yeast two-hybrid or co-immunoprecipitation analysis. Transgenic parasites in which the TgPCNA2 gene was disrupted displayed a slower growth rate in vitro; however, no difference in DNA polymerase activity, response to chemical mutagens, or recombinational frequency was observed in these mutant clones demonstrating that TgPCNA2 is non-essential in the tachyzoite developmental stage. Heterologous expression of TgPCNA1, but not TgPCNA2, was able to complement a POL30 cold-sensitive yeast strain suggesting that this isoform may serve as a major replisomal factor in T. gondii and is consistent with the failure to disrupt this gene in tachyzoites.