The fraction of chloroplast DNA transcribed at different stages of chloroplast development in Euglena gracilis was measured by RNA-DNA hybridization. Euglena cells were grown in the dark in a heterotrophic medium to stationary phase and then transferred to the light. Chloroplast development was monitored by the increase in the cellular chlorophyll content in the absence of cell division. Total cell RNA was isolated at various stages of chloroplast development, and hybridized in a vast excess to [125I]chloroplast DNA. The fraction of [125I]chloroplast DNA in the form of a duplex was monitored by chromatography on hydroxylapatite columns. The amount of RNA-DNA hybrid in the duplex mixture was determined by correcting for the contribution of DNA-DNA renaturation under the same conditions. The fraction of chloroplast DNA transcribed was calculated by multiplying by two the amount of single-stranded DNA in the form of an RNA-DNA hybrid. Prior to the initiation of chloroplast development (i.e., in dark grown cells) the fraction of chloroplast DNA represented as RNA transcripts in the cell is 0.53. As chloroplast development proceeds, the fraction of the chloroplast DNA transcribed decreases to 0.47. Experiments in which mixtures of various RNA samples were hybridized to the chloroplast DNA indicate that there is a small portion of chloroplast DNA transcribed at later stages of chloroplast development which is not represented as transcripts at the onset of chloroplast development. Melting properties of the RNA-DNA hybrids show that the RNA-DNA duplexes are slightly less stable than renatured [125I]chloroplast DNA.