Recombinant human sterol carrier protein 2 (SCP2) variants were generated by site-directed mutagenesis and expression in Escherichia coli. The ability of the variants to stimulate microsomal conversion of 7-dehydrocholesterol to cholesterol (sterol carrier activity) and to transfer cholesterol and phosphatidylcholine from donor small unilamellar vesicles to acceptor membranes (cholesterol and phosphatidylcholine transfer activities) was compared with wild-type recombinant SCP2. Our results indicate that all measured activities of recombinant human pre-SCP2 (including the 20-amino acid leader sequence) and mature SCP2 were similar. Expressed glutathione S-transferase fusion proteins (GST-SCP2 and GST-pre-SCP2) possessed considerable activity, suggesting that steric obstruction at the amino terminus causes only minor inactivation. The effect of progressive removal of peptides from the carboxyl terminus showed that amino acids between Lys100 and Asn104 are essential for SCP2 activity. This conclusion was substantiated by the observation that replacing Asn104 with Asp or Ile caused considerable inactivation, whereas replacing Met105 with Leu had almost no effect. Since N-ethylmaleimide is known to inhibit SCP2 activity, substitutions were also introduced in the vicinity of Cys71. Whereas Val71 and Ser71 variants possessed wild-type activity, replacing Asp70 with Asn almost completely abolished SCP2 activity. Further, the importance of residues located close to the amino terminus was indicated by complete inactivation of a 10-amino-terminal amino acid deletion mutant and by replacing Leu20 with Glu. Circular dichroism results showed that Leu20 and Asp70 may serve to stabilize the overall fold, whereas residue 104 appears to play a role in the specific lipid binding and/or transfer activity of SCP2.