Carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) activity were found in different Solanum lycopersicum fruit tissues, predominantly in the locular parenchyma and pericarp. The distribution of the CA and PEPC proteins in the tomato fruit tissues was examined by immunohistolocalization. CA and PEPC proteins were found in all fruit tissues examined as well as in the seeds. Three full length cDNA clones designated SlCA1, SlCA2 and SlCA3 coding for β-carbonic anhydrases (CA; EC 4.2.1.1) were identified and characterized from tomato fruit. SlCA1 and SlCA3 encode two putative cytosolic isoforms whereas SlCA2 encodes a putative plastidial isoform. Quantitative real time RT-PCR analysis revealed that accumulation of SlCA1 mRNA transcripts was detected in all examined tomato fruit tissues or organs, whereas SlCA2 gene transcripts were found in abundance in leaves. Stems also had SlCA2 transcripts, with transcript levels being higher in flowers than in stems. The SlCA3 gene transcripts were found only in the flowers and the roots. The SlPEPC1 and SlPEPC2 gene transcript levels in different fruit tissues of the tomato were also examined. Τhe possible role of CA isoforms in relation to PEPC in tomato fruit is discussed.