To study the effects of selection marker, promoter type, and copy number on heterologous expression in Aspergillus nidulans, strains were constructed with single- and multicopy plasmid integrations bearing a reporter gene (lacZ) under the control of either an inducible (alcA) or constitutive (gpdA) promoter and one of three Aspergillus nutritional marker genes (argB, trpC, or niaD). beta-Galactosidase activity in the transformants varied over three orders of magnitude, with the majority of levels in the range of 5x10(3)-1x10(4) U/mg. Significant differences in mean expression levels were found when comparing single-copy transformants with the same promoter but a different marker. Transformants with the argB marker had the highest average expression, similar to threefold over the trpC or niaD clones. For each promoter, maximal expression for the set was seen in the range of the single-copy clones, implying that increasing the copy number does not reliably increase expression in Aspergillus.