Plants have developed different strategies to adapt to various stress conditions including drought. In the present study the drought-induced changes in the actin filament (AFs) network was studied, for the first time, in two barley cultivars of contrasting drought tolerance level. Detached leaves of drought-tolerant (cv. 'CAM/B1/ CI') and drought-susceptible (cv. 'Maresi') cultivars were dried under controlled conditions. The water relations as well as the transcript accumulation of actin (ACT11), actin depolymerization factor (ADF1) and dehydrine (HVA1) encoding genes were studied using qRT-PCR. Quantitative (the relative fluorescence index; RFI) and qualitative drought-induced changes in AF cytoskeleton were observed following staining with phalloidin. It was noticed that tolerant cultivar was characterized with relative water content decreased during drought treatment which was accompanied by increase in HVA1 expression together with decrease in ACT11 and ADF1 transcripts accumulation induced by drought. In drought-susceptible cultivar the expressions of both ACT11 and ADF1 were slightly lower than those in the control. Drought triggered an extensive AF cytoskeleton reorganization within different types of leaf-blade cells. Remarkable changes in AF configuration and its increased amount (fluorescence intensity) were observed mainly in drought-tolerant cultivar. In addition, drought-induced changes in AFs were closely associated with chloroplasts. Those AFs probably controlled drought-induced intracellular chloroplast positioning in mesophyll. Based on the results obtained in the present study, the possible role of AF rearrangements in drought response is discussed.