We studied the influence of the method used in cultivating and preparing lymphocyte cultures on the quantitative and qualitative association patterns of the acrocentric chromosomes in the same individual. We compared the results obtained by macrocultures with medium TC 199 (method a), by macrocultures with McCoy’s medium 5A (method b) and by microcultures with McCoy’s medium 5A and 20% fetal calf serum (method c). These comparisons were made under two conditions: (1) fixation with glacial acetic acid/methanol 1:3 and (2) subsequent resuspension in 70% acetic acid. For cultures that were not resuspended, no marked differences could be observed in the quantitative association types {i.e., the number of associating chromosomes). As regards the qualitative association types (i.e., the relative frequency of participation of D and G chromosomes), however, an increasing deviation from the expected D-D:D-G:G-G ratio from method a to b to c, caused by an increase in G-G associations, could be demonstrated. In cultures resuspended in 70% acetic acid subsequent to the usual fixation process a significant increase in associations (primarily in associating G chromosomes) could be observed for all methods. Mechanical factors possibly responsible for these results are discussed.