Two β-CA genes, ptca1 and ptca2, were previously isolated from the marine diatom Phaeodactylum tricornutum and shown to be regulated by ambient [CO2] at the transcriptional levels. The product of ptca1, PtCA1, was also shown to be localized to the chloroplast as clumped particles. Both ptca1 and 2 encode putative N-terminal signal peptides of 19 amino acids. In the present study, the C-terminal region of the 46 amino acid presequence of PtCA1 (Pre46AA) was truncated to investigate the function of this region. DNA sequences, which encode the N-terminal 19, 23, 24, 34 or 44 polypeptides of Pre46AA, were fused with the sequence that encodes the mature protein, PtCA1. These constructs were fused with the enhanced-green fluorescent protein gene, egfp, and expressed in P. tricornutum. Fluorescent microscopy of expressed green fluorescent protein (GFP) showed that presequences of PtCA1 longer than the initial N-terminal 23 amino acids successfully sorted the PtCA1::GFP fusion into chloroplasts, whereas those shorter than 23 amino acids did not. These results indicate that the four amino acids after the endoplasmic reticulum signal peptide cleavage site are required as a chloroplast-targeting signal. Ala19 Phe20 is known to be the highly conserved transit motif among presequences of nuclear-encoded chloroplastic proteins in diatoms; however, the corresponding amino acid sequence deduced from the ptca2 was Ala19 Leu20. Substitution of Phe20 in PtCA1 with Leu did not alter the localization of PtCA1, indicating that the chloroplast-targeting motif possesses some alternative pattern and that PtCA2 could be sorted to chloroplast via a process similar to that of PtCA1. The ptca2 cDNA was isolated, fused at the 3′ terminus with the egfp and introduced into P. tricornutum cells. Expressed PtCA2::GFP fusion was clearly localized by chlorophyll fluorescence in the chloroplast of P. tricornutum as clumped particles, which was strikingly similar to the localization of PtCA1.