The effects of Cd, Cu, and Ni ions on chloroplast gene expression were studied in 7-day-old barley (Hordeum vulgare L.) seedlings. Toxicity of heavy metals (HM) was estimated from their effects on root and shoot growth and from the accumulation of HM in roots and leaves of seedlings grown in roll culture. Root growth was affected by HM to a higher extent than shoot growth. A statistically significant inhibition of root growth was observed at concentrations of all studied HM ≥100 μM. Leaf growth was inhibited by Cu and Ni at concentrations ≥400 μM, while the inhibitory effect of Cd on leaf growth was observed at concentrations ≥100 μM. The translocation of Cu from roots to shoots of barley seedlings proceeded slower than the accumulation of Cu in roots. Unlike Cu, Ni was actively accumulated by shoots in addition to its strong absorption by roots. Using the run-on transcription and RT-PCR (reverse transcription-polymerase chain reaction), we showed that HM at a concentration of 100 μM regulated the expression of individual chloroplast genes. The presence of either Cd, Cu, or Ni ions in the plant growth solution enhanced transcription of rpl16, rpl23, and rpl12 genes encoding ribosomal proteins, and of ndhA encoding the subunit 1 of plastid NADH-plastoquinone oxidoreductase. Transcription of sixteen other studied plastid genes was not affected by HM. The activation of transcription of the studied genes was not always accompanied by an increase in the transcript copy number. Northern blot analysis showed that Cd impaired the splicing of rpl16, a chloroplast gene that contains an intron. Thus, this study proves that HM regulate not only the transcriptional activity of chloroplast genes but also the splicing of rpl16 transcripts. © 2011 Pleiades Publishing, Ltd.