“Biological noise” is defined as functionally insignificant events that occur in living cells due to imperfect fidelity of biological processes. Distinguishing between biological function and biological noise is often difficult, and experiments to measure biological noise have not been performed. Here, we measure biological noise in yeast cells by analyzing chromatin structure and transcription of an 18 kb region of DNA whose sequence was randomly generated and hence is functionally irrelevant. Nucleosome occupancy on random-sequence DNA is comparable to that on yeast genomic DNA. However, nucleosome-depleted regions are much less frequent, and there are fewer well-positioned nucleosomes and shorter nucleosome arrays. Steady-state levels of RNAs expressed from random-sequence DNA are comparable to those of yeast mRNAs, although transcription and mRNA decay rates are at higher levels. Transcriptional initiation (5’ ends) from random-sequence DNA occurs at numerous sites at low levels, indicating very low intrinsic specificity of the Pol II machinery. In contrast, poly(A) profiles (relative levels and clustering of 3’ isoforms) of random-sequence RNAs are roughly comparable to those of endogenous yeast RNAs, which are restricted to 3’ untranslated regions. RNAs expressed from random-sequence DNA show higher cell-to-cell variability than RNAs expressed from yeast genomic DNA, suggesting that functional elements limit the variability among individual cells within a population. These observations indicate that transcriptional noise occurs at high levels in yeast, and they provide insight into how chromatin and transcription patterns arise from the evolved yeast genome. Overall design: 5'-Seq (5' isoforms or transcription start sites) of the strain harboring random-sequence artificial chromosome (ChrXVII).