Members of the photolyase/cryptochrome family are flavoproteins that share an extraordinary conserved core structure (photolyase homology region, PHR), but the presence of a carboxy-terminal extension is limited to the cryptochromes. Photolyases are DNA-repair enzymes that remove UV-light-induced lesions. Cryptochromes of plants and Drosophila act as circadian photoreceptors, involved in light entrainment of the biological clock. Using knockout mouse models, mammalian cryptochromes (mCRY1 and mCRY2) were identified as essential components of the clock machinery. Within the mammalian transcription-translation feedback loop generating rhythmic gene expression, mCRYs potently inhibit the transcription activity of the CLOCK/BMAL1 heterodimer and protect mPER2 from 26S-protesome-mediated degradation. By analyzing a set of mutant mCRY1 proteins and photolyase/mCRY1 chimeric proteins, we found that the carboxyl terminus has a determinant role in mCRY1 function by harboring distinguished domains involved in nuclear import and interactions with other clock proteins. Moreover, the carboxyl terminus must cross-talk with the PHR to establish full transcription repression capacity in mCRY1. We propose that the presence of the carboxyl terminus in cryptochromes, which varies in sequence composition among mammalian, Drosophila, and plant CRYs, is critical for their different functions and possibly contributed to shape the different architecture and biochemistry of the clock machineries in these organisms.