Room temperature (RT) microspectrofluorimetry in vivo of single cells has a great potential in photosynthesis studies. In order to get new information on RT chlorophyll fluorescence bands, we analyzed the spectra of Chlamydomonas reinhardtii mutants lacking fundamental proteins of the thylakoid membrane and spectra of photoinhibited WT cells. RT spectra of single living cells were characterized thorough derivative analyses and Gaussian deconvolution. The results obtained suggest that the dynamism in LHCII assembly could be sufficient to explain the variations in amplitudes of F680 (free LHCII), F694 (LHCII-PSII) and F702 (LHCII aggregates); F686 was assigned to the PSII core. Based on the revised assignments and on the variations observed, we discuss the meaning of the two fluorescence emission ratios F680/(F686 + F694) and F702/(F686 + F694), showing that these are sensitive parameters under moderate photoinhibition. In the most photoinhibited samples, the RT spectra tended to degenerate, showing characteristics of mutants that are partly depleted in PSII.