Background Keratinocyte (KC) apoptosis is believed to play an important role in the pathogenesis of spongiotic dermatitis, in particular for the formation of spongiosis. Objective To investigate changes in the expression level of the apoptosis regulatory proteins cleaved caspase-3, Fas, Bcl-2, nuclear factor kB (NF-kB) and p53 in skin samples of patients with spongiotic dermatitis. Patients and methods The present study included a total of 50 patients with spongiotic dermatitis and 10 healthy controls. Patients were classified into five groups (10 patients each): group A, atopic dermatitis; group B, allergic contact dermatitis; group C, irritant contact dermatitis; group D, nummular eczema; and group E, dyshidrotic eczema. Using immunohistochemistry, we investigated the expression of apoptotic regulatory proteins cleaved caspase-3, Fas, Bcl-2, NF-kB and p53 in skin biopsies taken from all patients and controls. Results The mean values of cleaved caspase-3 and Fas expression were statistically increased in patients with acute spongiotic dermatitis compared with the controls (Po0.001). In the spinous cell layer, cleaved caspase-3 and Fas expression was observed in all specimens taken from lesional skin with different intensities. The strongest positive staining was noticed in areas of spongiosis. Bcl-2 and NF-kB expression was absent or weak in suprabasal cells in the lesional skin with no statistically significant difference between the patient group and the control group (P= 0.74 and 0.38, respectively). p53 expression was absent or weak in suprabasal cells in the lesional skin, with no statistically significant difference between the patient group and the control group (P = 0.97). There was no difference in the mean expression of cleaved caspase-3, Fas, Bcl-2, NF-kB and p53 between different subsets of eczematous dermatitis (P = 0.60, 0.14, 0.68, 0.76 and 0.30, respectively). Conclusion Apoptosis of KC through an extrinsic pathway is the initiating event in the development of the epidermal pathology seen in spongiotic dermatitis. Most notably, KC apoptosis occurs in suprabasal cells, where spongiosis takes place.