- SSR (Strategic Scientific Reserve)
- Captain John Flynn (First appearance)
- Agent Miller
- Agent Johnson
- Agent Wilkes
- S.H.I.E.L.D. (Mentioned)
- Zodiac (First appearance)
- Captain America (Steve Rogers) (Only in flashback)
- Zodiac Virus (First appearance)
One year after Captain America's disappearance, Agent Peggy Carter of the SSR has been assigned to desk duties filing paperwork instead of working in the field. One night while alone in the office, the telephone rings, informing her of the location of the criminal organization known as Zodiac. She goes to the Zodiac base, takes out the Zodiac members single handed and is able to retrieve the serum/virus.
After the mission, Agent Flynn reprimands her for not going through the proper procedures. However the telephone rings, this time with Howard Stark on the other end. Stark informs Agent Flynn that Agent Carter will co-head the newly created S.H.I.E.L.D. and that Agent Flynn must personally assist Agent Carter packing her belongings.
After the call Dum Dum Dugan is poolside with Howard Stark, marvelling at two women wearing the newly created bikinis. Dugan asks Stark if he created them, to which Stark replies, "the French."
- Hayley Atwell as Agent Peggy Carter
- Dominic Cooper as Howard Stark
- Bradley Whitford as Agent Flynn
- Neal McDonough as Dum Dum Dugan
- According to Christopher Markus and Stephen McFeely, the ABC series Marvel's Agent Carter was meant to take place in the middle of the timeline of this short film.