The nuclear 2C DNA content of seven Pinus species and Pseudotsuga menziesii was determined by flow cytometric analysis. Conditions for nuclear isolation were adapted to optimise results for Pinus. To reduce endonuclease activity and the effect of high tannins and phenolics, 10 mM EDTA, 15 mM β-mercaptoethanol and 1% PVP-10 were added to the chopping buffer. The accessibility of propidium iodide, an intercalating stain, was improved by acid treatment of the nuclei with HCl for 1 min, leading to an increase of 10–30% in fluorescence. Using these optimised conditions, values for genome sizes of the eight species ranged from 38 to 53 pg per 2C nucleus. Little or no variation was observed between shoot apices or embryogenic cell cultures of P. radiata, except that one cell line and plants derived from it contained an additional 2 pg of DNA. Cytogenetic analysis showed this line to be trisomic, with 2n = 2x+1 = 25 chromosomes.