Fungal spores are a significant fraction of the atmospheric bioparticles (bioaerosols) and many species are capable of inducing the production of specific immunoglobulin E (IgE), aggravating the clinical symptoms of allergic respiratory diseases in sensitized individuals. The aim of this work was to evaluate the distribution of potentially allergenic Pleosporales spores in two locations with different urbanization indexes, characterizing its seasonal pattern. The seasonal distribution of several spore types belonging to the Pleosporales (Alternaria, Drechslera, Epicoccum, Paraphaeosphaeria, Pithomyces, Pleospora and Stemphylium) in Amares (rural area) and Porto (urban area) was continually studied from January 2005 to December of 2007, using Hirst-type volumetric spore traps. Alternaria was the most abundant fungal spore type found in the atmosphere of Amares and Porto. This fungal type, together with Drechslera, Epicoccum, Pithomyces and Stemphylium, was mainly present during summer. Nevertheless, Leptosphaeria, Pleospora and Venturia spores were detected during winter and spring, while Paraphaeosphaeria spores were also observed during summer and autumn. These different seasonal patterns were responsible for the expansion of the exposure period for the Alt a 1 allergen. The concentration of the studied spore types was higher in the rural area than in the urban one, with exception for Pleospora and Drechslera. According to the correlations with meteorological factors, the selected fungal spores can be divided into two groups: (i) Alternaria, Drechslera, Epicoccum, Pithomyces and Stemphylium presented positive correlations with temperature and negative correlations with relative humidity and rainfall; (ii) Leptosphaeria, Paraphaeosphaeria, Pleospora and Venturia presented a contrary behavior. Usually, the occurrence of the Alt a 1 allergen has been associated with the presence of airborne Alternaria spores; the present work follows the seasonal distribution of other fungal spore species known to contain this molecule. The widespread occurrence of Alt a 1 plays an important role in the incidence and aggravation of allergic disorders.