Photoperiod and circadian controls play crucial roles in the regulation of chloroplast biogenesis. To understand more about the regulation of this process, we compared the greening of the first leaves of wild type barley and two WHIRLY1 (WHY1)-deficient lines. Seedlings were grown in darkness for 4 days prior and then exposed to light at the beginning of the photoperiod on the 5th day or under standard photoperiod conditions. The accumulation of chlorophyll, as well plastid-encoded photosynthetic transcripts and proteins was delayed in the WHY1-deficient lines under standard photoperiod conditions because of defects in plastid gene expression, ribosomal processing and photosynthetic protein accumulation. The acquisition of full photosynthetic capacity was delayed by about 11 days in the first leaves and the newly forming leaves of the WHY1-deficient lines compared to the wild type. However, the light-dependent accumulation of pigments, transcripts and photosynthetic proteins was similar in all lines when etiolated seedlings were exposed to light. These results demonstrate that WHY1 is required for the integration of photoperiod-dependent signalling and chloroplast development in barley leaves.