In the unicellular flagellated green alga Chlamydomonas reinhardtii several processes are regulated by the circadian clock. To study circadian controlled processes, the cell’s clock is synchronized in a 12 h light–12 h dark cycle (LD12:12) before the cells are released into constant conditions of dim light and temperature. Under these free-running conditions circadian rhythms will continue with a period of about 24 h and cells can be harvested during specific time-points of subjective day and night. These cells were then used for isolating basic proteins by heparin-affinity chromatography, separating them on two-dimensional PAGE and comparing the amount of their expression at four different time-points of subjective day and night. Among 230 proteins, we could find two proteins whose expression level changed more than fourfold throughout the circadian cycle. These proteins were identified as a protein disulfide isomerase (PDI)-like protein and a tetratricopeptide repeat (TPR) protein by liquid-chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).