The mannose-binding lectin (MBL) pathway of complement activation is an important component of innate host defence. Numerous studies have described associations between the MBL genotype, MBL levels and disease susceptibility. However, genotyping and quantitative assays used in these studies have frequently been limited, and comprehensive data examining the interaction between structural and coding MBL genetic variants, MBL antigenic levels and MBL functional activity are lacking. Such data may be important for accurate planning and interpretation of studies of MBL and disease. This study has examined MBL in a cohort of 236 Australian blood donors. Five MBL promoter and coding single nucleotide polymorphisms were genotyped using polymerase chain reaction-sequence-specific priming (PCR-SSP). Plasma levels of MBL antigen were quantified using a double-antibody enzyme-linked immunosorbent assay (ELISA), and functional MBL levels were quantified using a mannan-binding assay. Activation of the complement pathway by MBL was measured in a C4-deposition assay. Significant associations were found between both coding and promoter polymorphisms and MBL antigenic and functional levels. There was significant correlation between the results of MBL double-antibody, mannan-binding and C4-deposition assays. Comprehensive MBL genotyping and functional MBL quantitation using mannan-binding and C4-deposition assays have the potential to be highly informative in MBL disease association studies.