A fully randomized sampling design was adopted to test whether pollen viability of Austrian pine (Pinus nigra Arnold) was impacted by NO 2 pollution. Spatial strata (500⁎500 m each) with high (41.9–44.6 µg m −3 ) and low (15.4–21.0 µg m −3 ) NO 2 were selected from a defined population in a small area (236.5 km 2 , <200 m range in elevation) in Northern Italy. Pollen viability was measured by means of the Tetrazolium (TTC) test. Analysis of variance by means of a generalised linear model showed that NO 2 was a significant factor (P=0.0425) affecting pollen viability. Within the treatment, no significant differences were detected among replicates. Within each replicate, sampling unit data were significantly different (P=0.000) and this suggested some improvement in the applied sampling design was needed. Pollen viability was significantly related to pollen germination (P<0.01) and tube length (P<0.01). This suggested a possible impact of NO 2 on the regeneration of Austrian pine in polluted environments.