CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro. In vivo,cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein. Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially. MATa cac1 cells exhibit an unusual response to α-factor: They arrest and form mating projections (shmoos) initially, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells. cac1 MATa HMLaHMRa strains do not form these shmoo clusters, indicating that derepression of HMLα causes the shmoo cluster phenotype in cac1 cells. When SIR3 is reintroduced intosir1 sir3 cells, HML remains derepressed indicating that SIR1 is required for the re-establishment of silencing atHML. In contrast, when SIR3 is reintroduced intocac1 sir3 cells, silencing is restored to HML,indicating that CAF-I is not required for the re-establishment of silencing. Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HML. Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin. MATa cac− sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively. We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin.