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Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (T EFF ) and central memory (T CM ) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced T EFF , T CM , as well as total IgG responses. T EFF and T CM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of T CM 'non-responders' observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Manish Sadarangani reports a relationship with GlaxoSmithKline that includes: funding grants. Manish Sadarangani reports a relationship with Merck & Co Inc that includes: funding grants. Manish Sadarangani reports a relationship with Pfizer that includes: funding grants. Manish Sadarangani reports a relationship with Sanofi Pasteur Inc that includes: funding grants. Manish Sadarangani reports a relationship with Seqirus Inc. that includes: funding grants. Manish Sadarangani reports a relationship with Symvivo that includes: funding grants. Manish Sadarangani reports a relationship with VBI Vaccines Inc that includes: funding grants.., (Copyright © 2021. Published by Elsevier Ltd.)